CRISPR technologies have progressed by leaps and bounds over the past decade, not only having a transformative effect on
biomedical research but also yielding new therapies that are poised to enter the clinic. In this review, I give an overview of (i)
the various CRISPR DNA-editing technologies, including standard nuclease gene editing, base editing, prime editing, and epigenome editing, (ii) their impact on cardiovascular basic science research, including animal models, human pluripotent stem
cell models, and functional screens, and (iii) emerging therapeutic applications for patients with cardiovascular diseases, focusing on the examples of Hypercholesterolemia, transthyretin amyloidosis, and Duchenne muscular dystrophy.
CRISPR : CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT
It is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity.
It forms the basis of a genome editing technology known as CRISPR-Cas9 that allows permanent modifications of genes within organisms.
CRISPR-Cas system consist of two key molecules that introduce a change into the DNA sequence 1. Cas 9 - act as molecular scissors 2. gRNA – guides Cas9 to the right part of the genome gRNA = crispr rRNA + tracrRNA
Prezi Link: https://prezi.com/q8lkxnmwk25-/untitled-prezi/?utm_campaign=share&utm_medium=copy
The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 (CRISPR-associated nuclease 9), a genome editing system adapted from the bacterial immune mechanism that is poised to transform genetic engineering by providing a simple, efficient and economical method to precisely manipulate the genome of any organism. Compared with zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), CRISPR/Cas9 is simpler with higher specificity and less toxicity. This RNA-guided nuclease (RGN)-based approach has been effectively used to induce targeted mutations(knock in or knock out) in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins.It has a wide variety of applications such as gene therapy, gene expression regulation, genome wide functional screening, virus resistance, transgenic animal production, site specific DNA integration etc. In the future CRISPR/Cas9 technology will play a significant role in innovating the life science research and industrial fields.
In this presentation, I talked about the new mRNA vaccine that is authorized for the prevention of coronavirus infection.
mRNA 1273 is developed by Moderna in the US and has shown almost 94% effectiveness
Have you considered that protein over-expression or inefficient mRNA knockdown may be masking physiological effects in your assays? Increasingly scientists are moving to endogenous gene-editing to characterise the function of their genes of interest.
Dr Chris Thorne from Cambridge Biotech Horizon Discovery discusses the ground breaking gene-editing technology CRISPR. The simplicity of experimental design has led to rapid adoption of the technology across the scientific community. However, challenges remain.
This Slidedeck focuses specifically on implementing CRISPR experiments, and explore a number of key considerations crucial to maximising chances of targeting success, whether your goal is to generate a knock-out or a knock-in. Chris also takes a look at some of the alternative uses of CRISPR, including sgRNA genome wide synthetic lethality screens.
The slides aim to support those researchers either planning to or already using CRISPR gene-editing in their lab. Horizon Discovery have also recently launched a program aimed specifically at academic cell biologists to promote the adoption of CRISPR by offering FREE CRISPR Reagents for knock-out cell line generation - more information available here. http://www.horizondiscovery.com/what-we-do/discovery-toolbox/genassist-crispr--raav-genome-editing-tools
CRISPR : CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT
It is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity.
It forms the basis of a genome editing technology known as CRISPR-Cas9 that allows permanent modifications of genes within organisms.
CRISPR-Cas system consist of two key molecules that introduce a change into the DNA sequence 1. Cas 9 - act as molecular scissors 2. gRNA – guides Cas9 to the right part of the genome gRNA = crispr rRNA + tracrRNA
Prezi Link: https://prezi.com/q8lkxnmwk25-/untitled-prezi/?utm_campaign=share&utm_medium=copy
The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 (CRISPR-associated nuclease 9), a genome editing system adapted from the bacterial immune mechanism that is poised to transform genetic engineering by providing a simple, efficient and economical method to precisely manipulate the genome of any organism. Compared with zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), CRISPR/Cas9 is simpler with higher specificity and less toxicity. This RNA-guided nuclease (RGN)-based approach has been effectively used to induce targeted mutations(knock in or knock out) in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins.It has a wide variety of applications such as gene therapy, gene expression regulation, genome wide functional screening, virus resistance, transgenic animal production, site specific DNA integration etc. In the future CRISPR/Cas9 technology will play a significant role in innovating the life science research and industrial fields.
In this presentation, I talked about the new mRNA vaccine that is authorized for the prevention of coronavirus infection.
mRNA 1273 is developed by Moderna in the US and has shown almost 94% effectiveness
Have you considered that protein over-expression or inefficient mRNA knockdown may be masking physiological effects in your assays? Increasingly scientists are moving to endogenous gene-editing to characterise the function of their genes of interest.
Dr Chris Thorne from Cambridge Biotech Horizon Discovery discusses the ground breaking gene-editing technology CRISPR. The simplicity of experimental design has led to rapid adoption of the technology across the scientific community. However, challenges remain.
This Slidedeck focuses specifically on implementing CRISPR experiments, and explore a number of key considerations crucial to maximising chances of targeting success, whether your goal is to generate a knock-out or a knock-in. Chris also takes a look at some of the alternative uses of CRISPR, including sgRNA genome wide synthetic lethality screens.
The slides aim to support those researchers either planning to or already using CRISPR gene-editing in their lab. Horizon Discovery have also recently launched a program aimed specifically at academic cell biologists to promote the adoption of CRISPR by offering FREE CRISPR Reagents for knock-out cell line generation - more information available here. http://www.horizondiscovery.com/what-we-do/discovery-toolbox/genassist-crispr--raav-genome-editing-tools
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.
An Introduction to Crispr Genome EditingChris Thorne
In this short presentation, I make a case for doing genome editing vs some of the approaches that have gone before, describe some of the tools available, and the focus on CRISPR-Cas9, what it is, where it's come from and how it works.
a brief description on the new emerging genome editing technology CRISPR-Cas9. this technique is making its place stronger and stronger day by day. and impossible things can be possible by this technique. and some main and famous names who discovered this technique.
CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.
Crispr-Cas9 system works on the concept of bacterial defence mechanism. The idea of which was replicated in eukaryotic cell in in- vitro condition by the researchers.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Akshay Deshmukh
clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria. Now CRISPR use as genome editing tool in different Plant Breeder to manipulate the DNA of the crop
Application of crispr in cancer therapykamran javidi
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9–DNA interactions, and associated conformational changes. The use of CRISPR–Cas9 as an RNA-programmable
DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)–CRISPR RNA (crRNA) structure
Introduction to CRISPR Cas9 technology. View in slide show after downloading for better viewing. Description is minimal, but it will be worth going through the slides that are full of pictures, if you have a minimal understanding of CRISPR.
Prepared in Oct 2015
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.
An Introduction to Crispr Genome EditingChris Thorne
In this short presentation, I make a case for doing genome editing vs some of the approaches that have gone before, describe some of the tools available, and the focus on CRISPR-Cas9, what it is, where it's come from and how it works.
a brief description on the new emerging genome editing technology CRISPR-Cas9. this technique is making its place stronger and stronger day by day. and impossible things can be possible by this technique. and some main and famous names who discovered this technique.
CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.
Crispr-Cas9 system works on the concept of bacterial defence mechanism. The idea of which was replicated in eukaryotic cell in in- vitro condition by the researchers.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Akshay Deshmukh
clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria. Now CRISPR use as genome editing tool in different Plant Breeder to manipulate the DNA of the crop
Application of crispr in cancer therapykamran javidi
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9–DNA interactions, and associated conformational changes. The use of CRISPR–Cas9 as an RNA-programmable
DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)–CRISPR RNA (crRNA) structure
Introduction to CRISPR Cas9 technology. View in slide show after downloading for better viewing. Description is minimal, but it will be worth going through the slides that are full of pictures, if you have a minimal understanding of CRISPR.
Prepared in Oct 2015
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
Describe the steps in Sanger DNA sequencing. Provide a high level des.pdffatoryoutlets
Consider the program: var s: int:= 1, i: int co i:= 1 to 2 rightarrow do true rightarrow (await s >
0 rightarrow s: = s - 1) (s:= s + 1) Above, S_i is a statement list that is assumed not to modify
shared variable s. Develop complete proof outlines for the two processes. Demonstrate that the
proofs of the processes are interference-free. Then use the proof outlines and the method of
Exclusion of Configurations (2.25) to show that S_1 and S_2 cannot execute at the same time
and that the program is deadlock-free. What scheduling policy is required to ensure that a
process delayed at its first await statement will eventually be able to proceed? Explain.
Solution
int i=1
do(i=1 to 2)
{
wait(s>0){
s=s-1
Si
}
s=s+1
}
to prove that it should be deadlock free it should support mutual exclusion,progress,bounded
waiting.
mutual exclusion:-
p1:
int i=1
do(i=1 to 2) //entry section
{
wait(s>0){ //critical section
s=s-1
Si
}
s=s+1
} //exit section
so while p1 is exexcuting p2 while s enters into critical section as we don\'t update any values so
mutual exclusion is possible and can be formed.
progress:-
when p1 is in non-critical section p2can execute in critical section.program gurantees progress.
bounded-waiting :-
suitable for ony countable no of procedures and processes.
as it satisfies all properties ....so it\'s deadlock free..
SBVRLDNACOMP:AN EFFECTIVE DNA SEQUENCE COMPRESSION ALGORITHMijcsa
There are plenty specific types of data which are needed to compress for easy storage and to reduce overall retrieval times. Moreover, compressed sequence can be used to understand similarities between biological sequences. DNA data compression challenge has become a major task for many researchers for the last few years as a result of exponential increase of produced sequences in gene databases. In this research paper we have attempt to develop an algorithm by self-reference bases; namely Single Base Variable Repeat Length DNA Compression (SBVRLDNAComp). There are a number of reference based compression methods but they are not satisfactory for forthcoming new species. SBVRLDNAComp is an optimal solution of the result obtained from small to long, uniform identical and non-identical string of nucleotides checked in four different ways. Both exact repetitive and non-repetitive bases are compressed by SBVRLDNAComp.The sound part of it is without any reference database BVRLDNAComp achieves 1.70 to 1.73 compression ratio α after testing on ten benchmark DNA sequences. The compressed file can be further compressed with standard tools (such as WinZip or WinRar) but even without this SBVRLDNAComp outperforms many standard DNA compression algorithms.
Gene editing application for cancer therapeuticsNur Farrah Dini
The application of TALENs as one of the gene editing tools in order to modify a specific targeted sites on a genome. This method shows a tremendous benefits especially in cancer research.
A gene mutation (myoo-TAY-shun) is a change in one or more genes. Some mutations can lead to genetic disorders or illnesses. A gene can mutate because of a change in one or more nucleotides of DNA, a change in many genes, loss of one or more genes, rearrangement of genes or whole chromosomes.
Wellens syndrome. Wellens syndrome (also referred to as LAD coronary T-wave syndrome) refers to an ECG pattern specific for critical stenosis of the proximal left anterior descending artery. The anomalies described occur in patients with recent anginal chest pain, and do not have chest pain when the ECG is recorded.
Congenital defects can put a strain on the heart, causing it to work harder. To stop your heart from getting weaker with this extra work, your doctor may try to treat you with medications. They are aimed at easing the burden on the heart muscle. You need to control your blood pressure if you have any type of heart problem.
Changing your lifestyle can help control and manage high blood pressure. Your health care provider may recommend that you make lifestyle changes including:
Eating a heart-healthy diet with less salt
Getting regular physical activity
Maintaining a healthy weight or losing weight
Limiting alcohol
Not smoking
Getting 7 to 9 hours of sleep daily
A post-splenectomy patient suffers from frequent infections due to capsulated bacteria like Streptococcus
pneumoniae, Hemophilus influenzae, and Neisseria meningitidis despite vaccination because of a lack of
memory B lymphocytes. Pacemaker implantation after splenectomy is less common. Our patient underwent
splenectomy for splenic rupture after a road traffic accident. He developed a complete heart block after
seven years, during which a dual-chamber pacemaker was implanted. However, he was operated on seven
times to treat the complication related to that pacemaker over a period of one year because of various
reasons, which have been shared in this case report. The clinical translation of this interesting observation
is that, though the pacemaker implantation procedure is a well-established procedure, the procedural
outcome is influenced by patient factors like the absence of a spleen, procedural factors like septic measures,
and device factors like the reuse of an already-used pacemaker or leads.
Transcatheter closure of patent ductus arteriosus (PDA) is feasible in low-birth-weight infants. A female baby was born prematurely with a birth weight of 924 g. She had a PDA measuring 3.7 mm. She was dependent on positive pressure ventilation for congestive heart failure in addition to the heart failure medications. She could not be discharged from the hospital even after 79 days of birth, and even though her weight reached 1.9 kg in the neonatal intensive care unit. We attempted to plug the PDA using an Amplatzer Piccolo Occluder, but the device failed to anchor. Then, the PDA was plugged using a 4-6 Amplatzer Duct Occluder using a 6-Fr sheath which was challenging.
Accidental misplacement of the limb lead electrodes is a common cause of ECG abnormality and may simulate pathology such as ectopic atrial rhythm, chamber enlargement or myocardial ischaemia and infarction
A Case of Device Closure of an Eccentric Atrial Septal Defect Using a Large D...Ramachandra Barik
Device closure of an eccentric atrial septal defect can be challenging and needs technical modifications to avoid unnecessary complications. Here, we present a case of a 45-year-old woman who underwent device closure of an eccentric defect with a large device. The patient developed pericardial effusion and left-sided pleural effusion due to injury to the junction of right atrium and superior vena cava because of the malalignment of the delivery sheath and left atrial disc before the device was pulled across the eccentric defect despite releasing the left atrial disc in the left atrium in place of the left pulmonary vein. These two serious complications were managed conservatively with close monitoring of the case during and after the procedure.
Trio of Rheumatic Mitral Stenosis, Right Posterior Septal Accessory Pathway a...Ramachandra Barik
A 57-year-old male presented with recurrent palpitations. He was diagnosed with rheumatic mitral stenosis, right posterior septal accessory pathway and atrial flutter. An electrophysiological study after percutaneous balloon mitral valvotomy showed that the palpitations were due to atrial flutter with right bundle branch aberrancy. The right posterior septal pathway was a bystander because it had a higher refractory period than the atrioventricular node.
Percutaneous balloon dilatation, first described by
Andreas Gruentzig in 1979, was initially performed
without the use of guidewires.1 The prototype
balloon catheter was developed as a double lumen
catheter (one lumen for pressure monitoring or
distal perfusion, the other lumen for balloon inflation/deflation) with a short fixed and atraumatic
guidewire at the tip. Indeed, initially the technique
involved advancing a rather rigid balloon catheter
freely without much torque control into a coronary
artery. Bends, tortuosities, angulations, bifurcations,
and eccentric lesions could hardly, if at all, be negotiated, resulting in a rather frustrating low procedural success rate whenever the initial limited
indications (proximal, short, concentric, noncalcified) were negated.2 Luck was almost as
important as expertise, not only for the operator,
but also for the patient. It is to the merit of
Simpson who, in 1982, introduced the novelty of
advancing the balloon catheter over a removable
guidewire, which had first been advanced in the
target vessel.3 This major technical improvement
resulted overnight in a notable increase in the procedural success rate. Guidewires have since evolved
into very sophisticated devices.
Optical coherence tomography-guided algorithm for percutaneous coronary intervention. Vessel diameter should be assessed using the external elastic lamina (EEL)-EEL diameter at the reference segments, and rounded down to select interventional devices (balloons, stents). If the EEL cannot be identified, luminal measures are used and rounded up to 0.5 mm larger for selection of the devices. Optical coherence tomography (OCT)-guided optimisation strategies post stent implantation per EEL-based diameter measurement and per lumen-based diameter measurement are shown. For instance, if the distal EEL-EEL diameter measures 3.2 mm×3.1 mm (i.e., the mean EEL-based diameter is 3.15 mm), this number is rounded down to the next available stent size and post-dilation balloon to be used at the distal segment. Thus, a 3.0 mm stent and non-compliant balloon diameter is selected. If the proximal EEL cannot be visualised, the mean lumen diameter should be used for device sizing. For instance, if the mean proximal lumen diameter measures 3.4 mm, this number is rounded up to the next available balloon diameter (within up to 0.5 mm larger) for post-dilation. MLA: minimal lumen area; MSA: minimal stent area;NC: non-compliant
Brugada syndrome (BrS) is an inherited cardiac disorder,
characterised by a typical ECG pattern and an increased
risk of arrhythmias and sudden cardiac death (SCD).
BrS is a challenging entity, in regard to diagnosis as
well as arrhythmia risk prediction and management.
Nowadays, asymptomatic patients represent the majority
of newly diagnosed patients with BrS, and its incidence
is expected to rise due to (genetic) family screening.
Progress in our understanding of the genetic and
molecular pathophysiology is limited by the absence
of a true gold standard, with consensus on its clinical
definition changing over time. Nevertheless, novel
insights continue to arise from detailed and in-depth
studies, including the complex genetic and molecular
basis. This includes the increasingly recognised
relevance of an underlying structural substrate. Risk
stratification in patients with BrS remains challenging,
particularly in those who are asymptomatic, but recent
studies have demonstrated the potential usefulness
of risk scores to identify patients at high risk of
arrhythmia and SCD. Development and validation of
a model that incorporates clinical and genetic factors,
comorbidities, age and gender, and environmental
aspects may facilitate improved prediction of disease
expressivity and arrhythmia/SCD risk, and potentially
guide patient management and therapy. This review
provides an update of the diagnosis, pathophysiology
and management of BrS, and discusses its future
perspectives.
The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.
The treatment of patients with advanced acute heart failure is still challenging.
Intra-aortic balloon pump (IABP) has widely been used in the management of
patients with cardiogenic shock. However, according to international guidelines, its
routinary use in patients with cardiogenic shock is not recommended. This recommendation is derived from the results of the IABP-SHOCK II trial, which demonstrated
that IABP does not reduce all-cause mortality in patients with acute myocardial infarction and cardiogenic shock. The present position paper, released by the Italian
Association of Hospital Cardiologists, reviews the available data derived from clinical
studies. It also provides practical recommendations for the optimal use of IABP in
the treatment of cardiogenic shock and advanced acute heart failure.
Left ventricular false tendons (LVFTs) are fibromuscular
structures, connecting the left ventricular
free wall or papillary muscle and the ventricular
septum.
There is some discussion about safety issues during
intense exercise in athletes with LVFTs, as these
bands have been associated with ventricular arrhythmias
and abnormal cardiac remodelling. However,
presence of LVFTs appears to be much more common
than previously noted as imaging techniques
have improved and the association between LVFTs
and abnormal remodelling could very well be explained
by better visibility in a dilated left ventricular
lumen.
Although LVFTsmay result in electrocardiographic abnormalities
and could form a substrate for ventricular
arrhythmias, it should be considered as a normal
anatomic variant. Persons with LVFTs do not appear
to have increased risk for ventricular arrhythmias or
sudden cardiac death.
The optimal management of bifurcation lesions has received significant interest in recent years and remains a matter of debate among the
interventional cardiology community. Bifurcation lesions are encountered in approximately 21% of percutaneous coronary intervention procedures
and are associated with an increased risk of major adverse cardiac events. The Medina classification has been developed in an attempt to
standardise the terminology when describing bifurcation lesions. The focus of this article is on the management of the Medina 0,0,1 lesion
(‘Medina 001’), an uncommon lesion encountered in <5% of all bifurcations. Technical considerations, management options and interventional
techniques relating to the Medina 001 lesion are discussed. In addition, current published data supporting the various proposed interventional
treatment strategies are examined in an attempt to delineate an evidence-based approach to this uncommon lesion.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
HOT NEW PRODUCT! BIG SALES FAST SHIPPING NOW FROM CHINA!! EU KU DB BK substit...GL Anaacs
Contact us if you are interested:
Email / Skype : kefaya1771@gmail.com
Threema: PXHY5PDH
New BATCH Ku !!! MUCH IN DEMAND FAST SALE EVERY BATCH HAPPY GOOD EFFECT BIG BATCH !
Contact me on Threema or skype to start big business!!
Hot-sale products:
NEW HOT EUTYLONE WHITE CRYSTAL!!
5cl-adba precursor (semi finished )
5cl-adba raw materials
ADBB precursor (semi finished )
ADBB raw materials
APVP powder
5fadb/4f-adb
Jwh018 / Jwh210
Eutylone crystal
Protonitazene (hydrochloride) CAS: 119276-01-6
Flubrotizolam CAS: 57801-95-3
Metonitazene CAS: 14680-51-4
Payment terms: Western Union,MoneyGram,Bitcoin or USDT.
Deliver Time: Usually 7-15days
Shipping method: FedEx, TNT, DHL,UPS etc.Our deliveries are 100% safe, fast, reliable and discreet.
Samples will be sent for your evaluation!If you are interested in, please contact me, let's talk details.
We specializes in exporting high quality Research chemical, medical intermediate, Pharmaceutical chemicals and so on. Products are exported to USA, Canada, France, Korea, Japan,Russia, Southeast Asia and other countries.
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
Anti ulcer drugs and their Advance pharmacology ||
Anti-ulcer drugs are medications used to prevent and treat ulcers in the stomach and upper part of the small intestine (duodenal ulcers). These ulcers are often caused by an imbalance between stomach acid and the mucosal lining, which protects the stomach lining.
||Scope: Overview of various classes of anti-ulcer drugs, their mechanisms of action, indications, side effects, and clinical considerations.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
2. 80 K. Musunuru
cells that are in the S phase or G2 phase and thus have double the usual
DNA content. HDR uses a homologous region of DNA—which can be
on the duplicate chromatid on the same chromosome, on a sister
chromosome, or on a heterologously introduced synthetic single-strand
oligonucleotide or double-strand DNA vector—as a repair template to
precisely replace the DNA around the site of the DSB. If the repair tem
plate carries an alteration flanked by homologous sequences, HDR can
stably incorporate the alteration into the genome (Figure 1). This phe
nomenon makes HDR best suited for the introduction or correction
of mutations, as well as the insertion of cassettes. Even in permissive
cells, HDR is typically much less efficient than NHEJ, and NHEJ can com
plicate efforts to yield cells with precise genotypes (e.g. HDR correcting
a heterozygous mutation on one allele but NHEJ knocking out the other
allele with an indel mutation).
Although there are a variety of programmable nucleases capable of ef
ficiently introducing DSBs at target genomic sites, by far the most widely
used are CRISPR–Cas systems.6
The prototypic system, Streptococcus
pyogenes CRISPR–Cas9 (SpCas9), is the most popular due to its ease
of use and its relatively high editing efficiencies. This system comprises
the Cas9 protein, which is a nuclease that cuts the two DNA strands
with two distinct catalytic domains and is often likened to molecular scis
sors, and a single guide RNA (gRNA) ≈100 nucleotides in length, which is
often likened to a GPS that directs Cas9 to the target genomic site.7
Cas9
tightly complexes with the gRNA and scans across double-strand DNA
molecules, pausing at protospacer-adjacent motifs (PAMs)—which in the
case of SpCas9 are NGG sequences (N = any base), occurring on aver
age at 1 out of 16 positions along any given DNA strand. If the first 20
nucleotides at the 5′
end of the gRNA (spacer sequence) match the 20
nucleotides on the same DNA strand as the PAM and just upstream of
the PAM (protospacer sequence, on the non-target DNA strand), the
spacer sequence will hybridize with the complementary sequence on
the other DNA strand (target strand) via Watson–Crick base pairing,
which activates Cas9 to make a blunt-end DSB three base pairs upstream
of the PAM. The Cas9/gRNA complex remains intact and can continue to
search for and cleave target sites—including the site of any accurately re
paired DSB that it had previously induced.
Besides SpCas9, there are a variety of other CRISPR–Cas9 systems
that have been adapted from various bacterial species, e.g.
Staphylococcus aureus CRISPR–Cas9 (SaCas9) which is often used due
to its smaller size compared with SpCas9.8
The various Cas9 proteins
have distinct PAM preferences, e.g. SaCas9 prefers NNGRRT sequences
(R = G or A). Protein engineering has yielded variants of SpCas9 and
SaCas9 with altered PAM preferences, including near-PAMless SpCas9
variants that have relaxed PAM requirements, greatly increasing the
range of genomic sites that can be targeted.9
A distinct Cas family, the
Cas12 proteins, are also available for use in standard nuclease gene
...........................................................................................................................................................................................
Table 1 Comparison of CRISPR technologies
Technology Types of intended
edits
Types of unintended edits Efficiency Size of gene
encoding the
editing protein
Components besides
editing protein
Standard nuclease gene
editing—
non-homologous
end-joining
Semi-random, small indel
mutations; precise
deletions if two guide
RNAs used
Small or large indel mutations;
loss of chromosomal
segments; chromosomal
rearrangements;
chromothripsis
(chromosome shattering)
Can be very high
(close to
100%
efficiency)
Medium (in some
cases can fit in
a single AAV
vector)
Guide RNA; two guide RNAs
for precise deletion
Standard nuclease gene
editing—
homology-directed
repair
Precise single-nucleotide
changes, insertions,
deletions, and any
combination thereof
Small or large indel mutations;
loss of chromosomal
segments; chromosomal
rearrangements;
chromothripsis
(chromosome shattering)
Typically low Medium (in some
cases can fit in
a single AAV
vector)
Guide RNA; single-strand or
double-strand DNA repair
template
(homology-directed
repair)
Base editing Single-nucleotide changes
(typically restricted to
transition mutations)
Small indel mutations; bystander
single-nucleotide changes at
target site; off-target DNA
deamination; off-target RNA
deamination
Can be very high
(close to
100%
efficiency)
Large (typically
requires
splitting across
multiple AAV
vectors)
Guide RNA
Prime editing Precise single-nucleotide
changes, insertions,
deletions, and any
combination thereof
Small indel mutations; off-target
prime edits; unknown
consequences of reverse
transcriptase activity
Typically low
(but
improving
with new
innovations)
Large (typically
requires
splitting across
multiple AAV
vectors)
Prime editing guide RNA
(pegRNA); two pegRNAs
for deletion or twin prime
editing
Epigenome editing No sequence changes;
changes to methylation
status and/or
chromatin
modifications
Off-target methylation and/or
chromatin changes; edits
might not be durable
Can be high Medium to large Guide RNA
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
3. CRISPR and cardiovascular diseases 81
editing; the best characterized are Cas12a/Cpf1, Cas12b/C2c1, and
Cas12e/CasX.10–12
In contrast to Cas9 proteins, Cas12 proteins use a
single catalytic domain to cut both DNA strands in a staggered configur
ation, tend to have PAM preferences weighted towards T-rich se
quences, use protospacer DNA sequences downstream of the PAMs,
and use spacer sequences at the 3′
ends of the gRNAs.
Standard nuclease gene-editing carries non-trivial risks of genotoxi
city. Even if DSBs occur at the desired target genomic sites, they can re
sult in large insertions or deletions that affect multiple genes, loss of
entire chromosomal segments, chromosomal rearrangements, and
even chromothripsis (chromosome shattering).13
This on-target collat
eral damage is increasingly coming to light as newer long-read
sequencing technologies are used to interrogate the targeted loci.
DSBs occurring at sites other than the intended target sites can result
in off-target mutagenesis, which carries the theoretical risk of disrupting
tumour suppressor genes or activating oncogenes and increasing the po
tential for carcinogenesis. A wide variety of techniques are available to
assess CRISPR–Cas systems for their propensity for off-target mutagen
esis, and when it occurs, it tends to be at genomic sites with a high de
gree of sequence similarity to the intended target site (limited number of
mismatches or bulges between the spacer and protospacer se
quences).14
Off-target mutagenesis can be mitigated via either Cas pro
tein engineering or modification of the guide RNA, though often with an
accompanying reduction of on-target editing efficiency.15–18
Figure 1 Standard nuclease gene editing with CRISPR–Cas9. CRISPR–Cas9 recognizes a genomic site defined by complementary base pairing of ≈20
nucleotides in the guide RNA (spacer) with the target DNA strand and the presence of an appropriate PAM in the non-target DNA strand. The arrows
indicate the sites of Cas9-mediated DNA cleavage to generate a double-strand break. Different outcomes occur depending on how many guide RNAs are
used, whether a custom-made DNA repair template is provided, and whether the double-strand break is repaired by NHEJ or HDR. Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
4. 82 K. Musunuru
2.2. Base editing
CRISPR–Cas9 lends itself to other modes of editing besides standard nu
clease gene editing because the DNA cleavage activity and the genomic tar
geting activity are separated into two molecules, the Cas9 protein, and the
gRNA, respectively. With partial or complete elimination of the cleavage
activity of Cas9—either by mutating one of the catalytic domains such
that Cas9 can only nick one DNA strand (nickase Cas9, or nCas9) or by
mutating both catalytic domains such that Cas9 cannot cut either DNA
strand (dead Cas9, or dCas9)—the Cas9/gRNA complex can still localize
to a desired target genomic site, allowing the complex to serve as a plat
form on which to tether additional enzymes at the target site.
In base editing, nCas9 is fused to either a cytidine deaminase domain
(cytosine base editing) or an adenosine deaminase domain (adenine base
editing).19,20
There are a wide variety of cytosine base editors, using
cytidine deaminase domains adapted from the many naturally occurring
proteins that act upon cytosine bases in single-strand DNA molecules.6
In contrast, there are relatively fewer adenine base editors; because
there are no naturally occurring deaminase proteins that act upon aden
ine bases in single-strand DNA molecules, these editors all rely on the
same group of adenosine deaminase domains that were evolved in the
laboratory from a single deaminase protein (TadA) that acts upon aden
ine bases in single-strand RNA molecules.20
Both cytosine and adenine base editors can catalyse site-specific,
single-nucleotide edits, and as such their action is often likened to a pencil
and eraser. When the nCas9/gRNA complex engages with the target
genomic site, the non-target DNA strand assumes a single-strand con
formation as the gRNA hybridizes with the target DNA strand (an
R-loop structure), which makes any bases within a certain window on
the non-target strand accessible to the deaminase domain fused to
Figure 2 Base editing. Tethering of a deaminase domain to nickase Cas9 (nCas9) can result in site-specific C-to-T or A-to-G edits on the non-target
strand without the need for double-strand breaks. The arrow indicates the site of a nick.
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
5. CRISPR and cardiovascular diseases 83
nCas9 (Figure 2). Cytosine base editors convert cytidine (C) to uridine
(U), and adenine base editors convert adenosine (A) to inosine (I).
Being non-standard in DNA, U is normally restored to C by the action
of the repair enzyme uracil-DNA glycosylase; fusion of yet another do
main to nCas9, a protein inhibitor of uracil-DNA glycosylase, prevents
this restoration. (Although I is non-standard as well, restoration to A is
slow, and there is no need to block an endogenous repair pathway for
adenine base editing.) The nickase activity of nCas9 is directed to the tar
get DNA strand only, which instigates a nick repair process that removes
a patch of nucleotides from that strand. Those nucleotides are replaced
via polymerase action that uses the complementary bases on the non-
target strand as the template, inserting an A opposite any U (which is
treated like a T), or a C opposite any I (which is treated like a guanosine,
or G). After the base editor has moved away from the site and nick repair
is complete, any U or I in the non-target strand is eventually replaced
with T or G via base excision and replacement by complementarity to
the opposing A or C on the target strand—completing the C–G to T–
A, or A–T to G–C, base pair change. (In unusual circumstances, a C–G
base pair can be edited to G–C by a cytosine base editor.21
)
Base editors are limited in the types of edits they can make (largely
C→T and A→G transition mutations) and the locations in which they
can make the edits (within the specific window on the non-target strand
dictated by the choice of base editor/deaminase and the positioning of
the PAM). There is also the possibility of undesirable bystander edits,
i.e. editing of bases other than the target base within the editing window.
Nonetheless, if the desired edit—whether a nonsense mutation or
splice site mutation to knock out a gene, or the correction of a disease-
causing mutation—is amenable to the action of a base editor, the editing
Figure 3 Prime editing. Tethering of an RT domain to nickase Cas9 (nCas9) and addition of a 3′
extension to the guide RNA to add a primer-binding
sequence (PBS) and RTT allows for RT to generate a variant-bearing DNA flap attached to the nicked non-target strand, which can ultimately replace the
corresponding portion of the original sequence. The arrow indicates the site of a nick.
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
6. 84 K. Musunuru
can potentially occur with close to 100% efficiency even in non-
proliferating cells. In such cases, base editing combines the high efficiency
of NHEJ and the precision of HDR while mitigating the disadvantages of
these repair modes of standard nuclease gene editing. Still, off-target
editing remains a concern. Although largely limited to single-nucleotide
changes—with minimal risk of large deletions or chromosomal rearran
gements due to base editing not relying on DSBs—stochastic off-target
editing from the deaminase domain interacting with single-stranded
DNA regions in a gRNA-independent fashion has been observed, almost
exclusively with cytosine base editors.22,23
As with standard nuclease
gene editing, off-target base editing can be mitigated by altering the pro
tein, whether in nCas9 or in the deaminase domain.
2.3. Prime editing
Prime editing, which is often likened to a word processor, offers the ad
vantages of base editing with respect to efficiency and precision while
overcoming the latter’s limitation of making only select single-nucleotide
changes.6,24
All possible nucleotide changes, as well as any small indel
mutations and even large deletions, lie within the scope of prime editing.
A version of nCas9 that nicks the non-target DNA strand (in contrast to
base editors, which nick the target DNA strand) is fused to a reverse
transcriptase (RT) (Figure 3). The gRNA has a 3′
extension that serves
two purposes—the distal end (primer-binding site, or PBS) hybridizes
with the non-target DNA strand upstream of the nick (i.e. starting
with the fourth nucleotide upstream of the PAM), and the intermediate
portion (reverse transcriptase template, or RTT) serves as a substrate
on which the RT can build a DNA flap directly attached to the non-
target DNA strand at the nick site. There is substantial flexibility with
respect to the length of the RTT and the mutations that can be incorpo
rated into the RTT—up to dozens of nucleotides and possibly even
longer—as well as positioning vis-à-vis a given PAM, offering an ex
panded targeting range compared to standard nuclease gene editing
and base editing. The prime editing gRNA (pegRNA) is vulnerable to
exonuclease action at its 3′
end, impairing the prime editing process,
and so the addition on the 3′
end of a structured RNA motif that is re
sistant to cleavage (engineered pegRNA, or epegRNA) can substantially
increase the efficiency of prime editing.25
Through a complex repair process, the mutation-bearing DNA flap
can displace and cause the excision of the local non-target DNA strand
downstream of the nick, followed by ligation that results in an intact
double-strand DNA in which the non-target strand has the desired mu
tation and the target strand has the original sequence. The mismatched
strands can be resolved in favour of either strand, but there are at least
two means by which to promote resolution in favour of the mutant
strand (resulting in the completion of prime editing). First, the same
nCas9-RT fusion protein can be used with a second, standard-length
gRNA with a spacer sequence that fosters a nick on the original strand;
nick repair then replaces the original nucleotides with nucleotides com
plementary to the mutant strand. Second, the cellular mismatch repair
(MMR) machinery tends to favour the original strand; accordingly, the
inhibition of MMR can increase the efficiency of prime editing, e.g. adding
a dominant-negative inhibitor of one of the MMR proteins to the
nCas9-RT protein.26
Similar to the use of two nucleases that introduce DSBs at sites flank
ing a region intended for deletion, prime editing with two pegRNAs can
engineer precise, large deletions.27,28
In what has been termed twin
prime editing, if the two pegRNAs generate flaps that contain comple
mentary sequences, they can be used to replace a DNA region with an
other DNA sequence encoded by the flaps.29
Although off-target mutagenesis with prime editing has been docu
mented, the full extent of off-target prime editing—in particular,
whether the use of RT poses any unexpected risks—remains to be clari
fied. If its safety proves to be favourable relative to other types of editing,
prime editing has the potential to become the preferred editing modality
for most applications in light of its versatility.
2.4 Epigenome editing
Epigenome editing is distinct from the other types of editing because it
does not involve any changes to DNA sequences. Rather, it modifies
gene expression by affecting how proteins interact with DNA se
quences. If a dCas9/gRNA complex is directed to a sequence in a
gene promoter or transcriptional enhancer (Figure 4), it will not directly
affect the DNA molecule but nonetheless can sterically interfere with
factors that normally interact with the sequence and thereby gene ex
pression (CRISPR interference).30
More potent knockdown can be
achieved if dCas9 is fused to a domain that actively suppresses gene ex
pression, such as the Krüppel-associated box (KRAB) domain, by modi
fying the local chromatin structure and thereby the accessibility of the
DNA sequence to the transcriptional machinery.31
Conversely, in
creased gene expression (CRISPR activation) can be achieved by fusing
dCas9 to a domain that acts as a transcriptional activator, such as VP16,
or by tethering activators to the gRNA by engineering RNA aptamers on
its 3′
end.32
These gene expression changes appear to be transient, in
that they persist only as long as the dCas9 fusion protein remains pre
sent at the site. More durable epigenome editing can be achieved if
dCas9 is fused to a methyltransferase or demethylase domain.33
Increased methylation in or near the promoter of a targeted gene, par
ticularly at cytosine bases in CpG dinucleotide sequences, typically re
sults in reduced gene expression, whereas decreased methylation
typically results in increased gene expression. Such methylation changes
have been observed to endure in cells through many divisions, and yet
the changes can be reversed by subsequently directing a dCas9 fusion
protein with the opposite effect to the same target genomic site.
3. Cardiovascular research
applications
3.1 Animal models
Perhaps the biggest impact of CRISPR on cardiovascular research—and
biomedical research generally—has been in facilitating the rapid gener
ation of genetically modified animal models. Standard nuclease gene edit
ing is well suited to the creation of knockout and knock-in mouse
models of disease in as little as three weeks (the length of mouse gesta
tion),34,35
so much so that it is supplanting the traditional means of gen
erating such models: in vitro modification of mouse embryonic stem cells
by homologous recombination, implantation into blastocysts to gener
ate chimeric mice, and a minimum of one round of breeding. NHEJ is
so efficient when SpCas9 is injected into zygotes (single-cell mouse em
bryos) that multiple genes can be fully knocked out simultaneously, sim
ply by co-injecting multiple gRNAs.34
The CRISPR components can be
delivered as DNA vectors, RNAs, or pre-assembled ribonucleoprotein
complexes. The introduction of indel mutations early in the coding se
quences of genes can result in relatively clean gene knockout, via the
dual mechanisms of severe truncation of protein products and
nonsense-mediated decay of messenger RNAs. NHEJ also permits dis
ruption of regulatory elements and, when using two gRNAs, precise de
letions in the genomes of zygotes.
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
7. CRISPR and cardiovascular diseases 85
Not unexpectedly, HDR to precisely insert a mutation, tag, or report
er upon co-injection of SpCas9, gRNA, and a synthetic DNA repair tem
plate into mouse zygotes is typically much less efficient than NHEJ
mutagenesis. Nonetheless, just one correctly edited allele in the zygote,
yielding a heterozygous mouse, permits the breeding of a large colony of
mice with the desired genotype. Moreover, if the other allele in the zyg
ote undergoes undesired NHEJ editing, that allele can be bred out of the
colony; the same is true of any off-target edit unless it is close enough on
the same chromosome to co-segregate with the desired edit. It is pos
sible to simultaneously introduce multiple alterations (e.g. two loxP sites
flanking a portion of a gene) by co-injecting multiple gRNAs and multiple
repair templates, though success is infrequent.36
Alternatively, multiple
mutations can be introduced serially: one round of HDR to insert the
first mutation, followed by breeding of enough correctly targeted
mice to obtain zygotes for a second round of HDR to insert the second
mutation, etc.37
Along the same lines, it is quite feasible to start with zy
gotes from mice of any genetic background and use CRISPR to add add
itional mutations to that background.
A decisive advantage of zygote editing is that it can be applied widely
across species, in principle allowing for any kind of animal to serve as a
disease model. This versatility is especially pertinent to cardiovascular
diseases, which are often poorly modelled in mice. Models that better
phenocopy human diseases include rats, rabbits, pigs, and non-human
primates, all of which have proven to be quite amenable to CRISPR
modification. In one example, CRISPR nuclease editing to knock out
the DMD gene generated a pig model of Duchenne muscular dystrophy
that displayed skeletal and cardiac muscle defects.38
In another example,
CRISPR nuclease editing to knock out the SAP130 gene, which encodes a
Figure 4 Epigenome editing. The use of catalytically dCas9, with tethering of a regulatory domain or enzyme to either Cas9 or the guide RNA (or both),
can introduce chromatin changes or DNA methylation changes that result in transcriptional activation or interference with a target gene’s expression with
out alteration of the DNA sequence.
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
8. 86 K. Musunuru
chromatin modifier previously linked to congenital heart disease, yielded
pigs with tricuspid valve dysplasia or atresia.39
An alternative approach to zygote editing for the purpose of disease
modelling is somatic editing, in which the editing is undertaken in living
animals. Viral vectors, particularly adeno-associated virus (AAV) vectors,
have proven to be effective in delivering CRISPR–Cas systems into or
gans such as the liver and the heart. A disadvantage of AAV vectors is
the limited cargo size, roughly 4.7 kb, which cannot easily accommodate
the gene encoding SpCas9 (≈4.2 kb, not including the requisite pro
moter and polyadenylation sequence) and a gRNA expression cassette
(≈0.5 kb). One workaround is the use of a smaller Cas9 isoform such as
SaCas9 (≈3.3 kb) so that the entire CRISPR–Cas system can fit into a
single AAV vector.8
Another strategy is to split up the components
among multiple AAV vectors. SpCas9, as well as the larger fusion pro
teins used for base editing or prime editing, can be split into two parts
that upon delivery into cells with dual AAV vectors can spontaneously
assemble into a working protein via intein-mediated splicing (split-intein
strategy).40
Yet another strategy is to use an animal model that already
has the gene encoding the Cas protein within its cells—e.g. Cas9 trans
genic mice—allowing for straightforward delivery of a gRNA or multiple
gRNAs into the target cells with a single AAV vector.41
Finally, non-viral
approaches such as lipid nanoparticles (LNPs)42
and virus-like particles43
have the potential to deliver CRISPR–Cas systems as RNAs and ribonu
cleoprotein complexes, respectively, with less constraint on size, though
their organ-targeting range remains more limited than that of AAV
vectors.
In one example of somatic editing for disease modelling, CRIPSR nu
clease editing in a cardiac-specific Cas9 transgenic mouse sufficiently
knocked out the Myh6 gene in the heart to cause hypertrophic cardio
myopathy.44
In another example, CRISPR nuclease editing in a condi
tional Cas9 transgenic mouse model individually knocked out each of
nine different genes in the heart and demonstrated functions for
junctophilin-2 and ryanodine receptor-2 in T-tubule stabilization and
maturation, respectively.45
3.2 Human pluripotent stem cell models
Human pluripotent stem cells, particularly induced pluripotent stem
cells (iPSCs), are increasingly being used as substitutes or complements
to animal models of cardiovascular diseases, as they provide a ready
source of human cells for the study of disease phenotypes.46
iPSCs
have several advantages over immortalized or transformed cultured
cell lines: they have normal human karyotypes, they are genetically
matched to the person of origin (who is often selected due to their dis
ease status), and they can be differentiated into a variety of cell types
relevant to cardiovascular diseases, including cardiomyocytes, vascular
endothelial cells, smooth muscle cells, hepatocytes, and macrophages.
When one is studying the impact of a genetic variant on disease, an
important consideration for any experiments involving patient-derived
iPSCs is the choice of control iPSCs used to determine whether the for
mer have meaningful phenotypes. Simply using iPSCs from healthy indi
viduals as comparators render any apparent phenotypes susceptible to a
variety of confounders—differences in genetic background, sex, ethni
city, epigenetics, pluripotency, capacity to differentiate into the desired
cell type, method of derivation, and other characteristics. CRISPR editing
offers a means to reduce these confounders and isolate the effects of the
variant in question, by removing the variant while leaving the iPSCs
otherwise unchanged (isogenic cells). Alternatively, editing can introduce
a variant into wild-type iPSCs, which can be useful when iPSCs are not
available from a patient with that variant. Furthermore, editing can
generate an allelic series of variant cell lines that are all on the same gen
etic background, allowing for a rigorous comparison of the relative ef
fects of the variants.
As with animal models, edited iPSC lines have been used to study a
broad range of cardiovascular traits and diseases including cardiomyop
athies, lipid metabolism, vascular disorders, valvular disease, and arrhyth
mia disorders.46
Besides assessing the effects of pathogenic variants on
disease phenotypes, iPSCs have been used to dissect molecular mechan
isms by relating gene function to phenotypes (e.g. gene knockout or
knockdown), coding variants to protein function, and non-coding var
iants to gene expression.
Beyond their use in modelling studies to dissect disease mechanisms,
iPSCs are increasingly being employed for translational purposes.
Isogenic iPSCs with and without variants of uncertain significance, iden
tified in patients known or suspected to have inherited cardiovascular
diseases, can clarify whether the variants are pathogenic or benign.47,48
In one example, for a 65-year-old woman with hypertrophic cardiomy
opathy who was found to have a variant of uncertain significance in
TNNT2, isogenic iPSCs with the variant were generated and phenotyped
in ,3 months, in between the patient’s first and second genetics clinic
visits. The work ascertained the variant to be a likely benign variant,
which was communicated to the patient and her provider and incorpo
rated into the management plan in real time (e.g. advising against cascade
genetic testing for the variant in family members).48
An important caveat
is that the prognostic capabilities of iPSCs for variant classification re
main unproven. A different translational application of iPSCs is to gener
ate somatic cell types for which obtaining primary cells is prohibitive, e.g.
cardiomyocytes, for use as a test platform in which to assess the efficacy
and safety of CRISPR–Cas systems that are ultimately intended to be de
ployed for therapeutic uses in patients.
The most commonly used strategy for introducing or removing var
iants in iPSCs is standard nuclease gene editing with HDR, typically via
transfection or electroporation of a CRISPR–Cas system along with a
single-strand DNA oligonucleotide to serve as a repair template, often
followed by fluorescence-activated cell sorting (FACS) or transient anti
biotic selection to enrich for cells receiving the editing components (par
ticularly if using DNA vectors to deliver the CRISPR–Cas system), and
then clonal outgrowth of single cells. This approach is notable for its ex
treme locus-to-locus variability in efficacy. In some cases, the proportion
of correctly edited clones may exceed 10%, but in many cases, the pro
portion is ,1% and often much ,1%, requiring the screening of hun
dreds of clones. This phenomenon can present a substantial challenge
given that rigorous iPSC-based studies will ideally use multiple clones
of each genotype, to mitigate the consequences of an off-target edit in
any given clone or of stochastic clone-to-clone heterogeneity. The use
of a DNA vector with an embedded antibiotic resistance cassette, close
to the variant, as the repair template allows for sustained antibiotic se
lection that substantially increases the reliability and efficiency of editing;
but this strategy requires a subsequent manipulation to remove the cas
sette from the genome, typically with a scarless approach such as the
piggyBac transposon system.49
3.3 Genome-wide or targeted functional
screens
A cardinal advantage of CRISPR–Cas systems is that they can target nu
merous genomic sites in parallel. With SpCas9-based systems—
whether standard nuclease gene editing, base editing, prime editing, or
epigenome editing—targeting the protein to a different site is simply a
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
9. CRISPR and cardiovascular diseases 87
matter of altering the first 20 nucleotides of the gRNA. Accordingly, it is
straightforward to clone a pool of as many as hundreds of thousands of
short oligonucleotides into a common plasmid backbone in order to
generate a library of gRNA-expressing vectors. In principle, this permits
the generation of lentiviral or AAV pools for infection of cells in vitro or in
vivo to screen for the effects of individual or combinatorial gene perturb
ation on a phenotype of interest. An individual academic laboratory can
readily make a library with multiple gRNAs targeting each of the ≈20
000 genes in the human genome or, alternatively, make a small library
that screens a defined subset of genes, e.g. those that encode kinases
or those that encode transcription factors.
Genome-wide and other large-scale CRISPR screens using standard
nuclease gene editing, CRISPR interference, CRISPR activation, and
base editing have become well-established methodologies.50
Standard
nuclease gene editing and CRISPR interference are complementary ap
proaches; gene knockout results in null alleles that can unmask other
wise hidden phenotypes but can disrupt essential genes, whereas
partial gene knockdown can reveal subtler phenotypes. CRISPR activa
tion provides a complementary approach to cDNA library screening
and overcomes the limitation that large genes are difficult to overex
press as cDNAs. Base editing can lend itself to gene knockout but can
also model the effects of a spectrum of variants ranging from dominant-
negative activity to gain of function. Designing a screen is a nuanced de
cision that entails consideration of a variety of factors: the type of editor;
the size and comprehensiveness of the library to be used; the cell type to
be studied; whether the targeted cells already stably express the Cas
protein, or whether the Cas and gRNA components are both delivered
via viral vectors; whether to perform the screen in vitro (much more
commonly done) or in vivo; the phenotype to be screened (typically a
self-selecting phenotype, like cell proliferation or survival upon exposure
to a drug, or a phenotype that can be identified through a marker or anti
body, permitting FACS); and whether to focus on positive selection
(identifying gRNAs enriched in the cells selected for analysis) or negative
selection (identifying gRNAs depleted in the cells) or both. Finally, any
hits that emerge from the primary screen need to be subjected either
to secondary screening or individual experimentation—ideally with
gRNAs distinct from the ones used for the primary screen—for valid
ation as true-positive findings.
One set of examples of productive CRISPR screens involved the
identification of modifiers of LDL cholesterol metabolism, particularly
the action of the LDL receptor—which resides at the cell surface and
moves LDL particles out of the bloodstream and into the cell—and
the proprotein convertase subtilisin/kexin type 9 (PCSK9) protein—
which acts as an antagonist to LDL receptor by binding to it at the
cell surface and causing it to be internalized and degraded in the cell.
In the first study, a genome-wide CRISPR nuclease screen in HEK
293T cells stably expressing PCSK9 fused to green fluorescent protein
sought to identify modifiers of PCSK9 secretion.51
The most enriched
gRNAs in FACS-isolated cells with the highest amount of internal fluor
escence (inhibited PCSK9 secretion) all targeted the SURF4 gene, an
endoplasmic reticulum cargo receptor. Further investigation revealed
that SURF4 directly interacts with PCSK9 in the early secretory path
way, promoting the efficient export and secretion of PCSK9. Three sub
sequent studies sought to discover novel modifiers of cellular LDL
uptake. One study used genome-wide CRISPR nuclease screening in
HuH-7 human hepatoma cells and targeted secondary screening in
HuH-7 and HepG2 human hepatoma cells to identify enriched
gRNAs in cells with very high or very low uptake of fluorescently con
jugated LDL particles; among the novel hits were genes encoding
components of the exocyst, an octameric protein complex involved
in vesicular trafficking.52
Another study used genome-wide CRISPR nu
clease screening in HepG2 cells to identify enriched gRNAs in cells with
very low uptake of fluorescently conjugated LDL particles; follow-up in
dividual experimentation confirmed three hits—SYNRG, C4orf33, and
TAGLN.53
Subsequent investigation found transgelin (the protein prod
uct of TAGLN), which is an actin-binding protein, to be involved in LDL
receptor endocytosis. The third study used genome-wide CRISPR inter
ference (dCas9-KRAB) screening in HepG2 cells to identify enriched
gRNAs in FACS-isolated cells with high or low surface LDL receptor ex
pression; follow-up individual experimentation confirmed many hits and
focused attention on the top-performing hit, CSDE1, which was found
to promote the degradation of the LDLR messenger RNA.54
The com
plementary findings of these various studies demonstrate the benefit of
using orthogonal CRISPR-based approaches to answer similar biological
questions.
In one example of a targeted in vivo functional screen, AAV vectors
with gRNAs against a set of 2444 genes focused on transcription factors
and epigenetic modifiers were administered as a pool into neonatal
transgenic mice harbouring both SpCas9 and a Myh7-yellow fluorescent
protein reporter acting as a marker of cardiomyocyte maturation.55
After 1 month, FACS-isolated cardiomyocytes with high reporter ex
pression were assessed for either enrichment or depletion of gRNAs.
After individual experimentation with the top novel hits, the related
Rnf20 and Rnf40 genes emerged as positive regulators of cardiomyocyte
maturation. RNF20 and RNF40 are components of a ubiquitin ligase
complex that monoubiquitinates histone H2B on lysine 120, an epigen
etic mark that controls dynamic changes in gene expression required for
maturation.
4. Therapeutic applications
4.1 Hypercholesterolaemia
Therapeutic CRISPR editing entails two approaches: ex vivo editing, in
which a patient’s cells are extracted, edited outside of the body, and
transplanted back into the body; and in vivo somatic editing, in which
the editing tool is administered directly into the body. As a practical mat
ter, it is unlikely that for the cell types most relevant to cardiovascular
diseases—cardiomyocytes, hepatocytes, vascular cells, etc.—ex vivo
editing and re-transplantation will be a viable approach for the foresee
able future. As such, this section focuses on emerging in vivo somatic
editing applications that target the liver or the heart. Three diseases,
in particular, stand out as eminently addressable by CRISPR editing in hu
man patients, with clinical trials underway or on the horizon: hyperchol
esterolaemia, transthyretin amyloidosis, and Duchenne muscular
dystrophy.
The best validated genetic targets for the treatment of hypercholes
terolaemia are the gene encoding the aforementioned PCSK9 protein
and the angiopoietin-like 3 (ANGPTL3) gene.56
For either gene, single
loss-of-function variants result in reduced LDL cholesterol levels and
in protection against coronary heart disease without any serious adverse
health consequences, and double loss-of-function variants (complete
gene knockout) have been observed in healthy individuals. Both genes
are largely expressed in the hepatocytes, and the protein products are
secreted into the bloodstream. Monoclonal antibodies against either
protein result in substantially reduced LDL cholesterol levels—up to
≈60%—in hypercholesterolaemic patients and have been approved
for medical use, and short-interfering RNAs and antisense
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
10. 88 K. Musunuru
oligonucleotides are in clinical trials. A limitation common to these treat
ments as well as to the mainstay of lipid-lowering medications, the sta
tins, is that they must be taken repeatedly for the lifetime (whether
every day, every few weeks, or every few months) in order to achieve
the full therapeutic benefit. CRISPR editing of either PCSK9 or
ANGPTL3 in the hepatocytes would potentially confer the same thera
peutic benefit with a single course of treatment, i.e. a ‘one-and-done’ so
lution to hypercholesterolaemia, due to the permanence of changes to
the DNA sequence.
In an early study, an adenoviral vector encoding SpCas9 and a gRNA
targeting a sequence in exon 1 of the mouse Pcsk9 gene was adminis
tered to mice in order to knock out Pcsk9 alleles in hepatocytes via
NHEJ.57
Although adenoviral vectors are generally not used for genetic
therapies due to the risk of life-threatening immune responses, one was
used in this proof-of-concept study to accommodate the large size of
SpCas9. Several days after receiving the treatment, the mice had
.50% whole-liver editing at the Pcsk9 target site. Consistent with
NHEJ repair, the most common edits were 1 or 2 bp deletions or inser
tions, although deletions as large as dozens of base pairs were evident.
Consistent with the high degree of editing, there were reductions in
blood PCSK9 protein levels of ≈90% and of blood cholesterol levels
of ≈40%, nearly as large as cholesterol reductions observed in germline
Pcsk9 knockout mice. Screening of a handful of candidate off-target sites,
chosen by high sequence similarity to the Pcsk9 target site, showed no
evidence of editing. A subsequent study using an adenoviral vector
with SpCas9 and the same Pcsk9 gRNA confirmed the therapeutic effect
on cholesterol levels and performed a much more rigorous assessment
of off-target editing, using a two-stage approach.58
First, a biochemical
technique called CIRCLE-seq—in which circularized mouse genomic
DNA fragments were mixed with SpCas9 protein and the Pcsk9
gRNA in vitro, followed by next-generation sequencing to identify any
DNA fragments that were linearized by SpCas9 cleavage—identified
182 candidate off-target sites. Second, PCR amplification of each of
the candidate sites from liver genomic DNA samples obtained from
the treated mice, followed by deep next-generation sequencing, showed
no evidence of off-target editing in vivo.
While these studies established the efficacy and off-target safety of
Pcsk9 editing in mice, the findings have limited relevance to a potential
PCSK9-editing therapy in human patients. First, the DNA sequences of
the mouse Pcsk9 gene and the human PCSK9 gene have substantial dif
ferences, and so even if the same exon 1 site were to be targeted in hu
man hepatocytes, the gRNA spacer sequence would be distinct and
might not support the same efficacy of editing. Second, the favourable
off-target profiling observed in the mouse genome might not hold up
in the human genome, due to the profound differences between the
two genomes. An independent assessment would need to be performed
in human hepatocytes. Third, the significant physiological differences be
tween mouse and human hepatocytes could mean that even if the same
gRNA were being used to target the same target site, the editing out
comes could very well differ between the two species. To establish a
preclinical model system in which to better test a human-directed ther
apy, chimeric liver-humanized mice—a xenograft model in which the
mouse hepatocytes are replaced with transplanted primary human he
patocytes that engraft in the liver—received an adenoviral vector encod
ing SpCas9 and a gRNA targeting a sequence in exon 1 of the human
PCSK9 gene.59
There was ≈50% editing of the PCSK9 gene within the hu
man hepatocytes in the liver, predominantly small indel mutations, and a
reduction of human PCSK9 protein levels in the blood of ≈50%.
Screening of a handful of candidate off-target sites showed no evidence
of editing. A subsequent study performed a much more rigorous assess
ment of off-target editing in the liver-humanized mice treated with the
adenoviral vector.60
First, a biochemical technique called ONE-seq—
in which tens of thousands of synthetic DNA oligonucleotides matching
genomic sites selected for their high sequence similarity to the PCSK9
target site were mixed with SpCas9 protein and the PCSK9 gRNA in vitro,
followed by next-generation sequencing to identify any cleaved DNA
fragments—rank-ordered all of the tested sequences by cleavage activ
ity. Second, PCR amplification of each of the 40 top-ranked sites from
liver genomic DNA samples obtained from the treated mice, followed
by deep next-generation sequencing, showed off-target editing at four
of the sites in vivo, arguing against further development of the human-
specific gRNA used in these studies into a therapy.
A separate set of studies turned to delivery methods more amenable
for use in human therapeutics than adenoviral vectors. Taking advantage
of the small size of SaCas9 compared with SpCas9, one study adminis
tered to mice an AAV vector encoding SaCas9 along with either of two
gRNAs targeting the mouse Pcsk9 gene, with the intent of using NHEJ to
disrupt the gene in the liver.8
Similar to the aforementioned adenoviral
mouse studies, AAV treatment resulted in 40–50% whole-liver Pcsk9
editing, with small indel mutations being the most frequent editing out
come, and reductions of blood PCSK9 protein levels of .90% and of
blood cholesterol levels of ≈40%. There was no evidence of off-target
editing at a handful of candidate sites in liver genomic DNA samples
from treated mice. The success of AAV-mediated somatic nuclease
gene editing in mice was followed by an exploration of non-viral meth
ods to deliver CRISPR–Cas systems into the mouse liver. One study seri
ally injected LNP formulations with in vitro transcribed SpCas9
messenger RNA or a chemically synthesized gRNA targeting a sequence
in the mouse Pcsk9 gene.61
The LNP treatments resulted in reductions
of PCSK9 protein levels in the liver of 40%–50%. A more comprehensive
study co-injected LNP formulations with either the SpCas9 messenger
RNA or a mix of two gRNAs targeting distinct sequences in Pcsk9,
each of which had chemical modifications intended to enhance RNA sta
bility in vivo.42
The LNP treatment resulted in .80% whole-liver editing
of the gene, with NHEJ-mediated deletion between the two gRNA tar
get sites being the most frequent editing outcome. The editing was ac
companied by an absence of detectable blood PCSK9 protein and a
reduction of blood cholesterol levels of ≈40%. No Pcsk9 editing was evi
dent in the lungs or spleen, suggesting that the LNPs preferentially tar
geted the liver, the Pcsk9 locus was accessible to nuclease action only in
liver cells, or both. A subsequent study treated mice with a single LNP
formulation with both SpCas9 messenger RNA and a gRNA targeting
a sequence in the mouse Angptl3 gene and achieved ≈40% whole-liver
Angptl3 editing and a reduction of blood ANGPTL3 protein levels of
≈65%.62
An even more recent study used engineered virus-like particles
to deliver CRISPR ribonucleoproteins into the mouse liver, resulting in
≈63% whole-liver Pcsk9 editing and a reduction of blood protein
PCSK9 levels of ≈78%.43
Yet another series of studies explored the use of CRISPR technolo
gies besides standard nuclease gene editing. Cytosine base editors can
knock out genes either through the direct introduction of nonsense mu
tations via C-to-T changes on the sense strand (CAG→TAG,
CAA→TAA, CGA→TGA) or C-to-T changes on the antisense strand
(TGG→TAG, TGG→TGA, TGG→TAA); the disruption of the start co
don (ATG→ATA); or the disruption of a canonical splice donor
(GT→AT) or splice acceptor (AG→AA). In contrast, adenine base edi
tors are restricted to disruption of the start codon (ATG→GTG,
ATG→ACG), a splice donor (GT→GC), or a splice acceptor
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
11. CRISPR and cardiovascular diseases 89
(AG→GG). One study administered to mice an adenoviral vector en
coding a cytosine base editor and a gRNA targeting the Pcsk9
tryptophan-159 codon (TGG), with the intent to introduce nonsense
mutations.63
The base-editing treatment resulted in ≈30% whole-liver
editing of the gene, with nonsense mutations being the predominant
editing outcome, although low levels of bystander missense mutations
and indel mutations were evident as well. There were reductions of
blood PCSK9 protein levels of ≈60% and of blood cholesterol levels
of ≈30%. A separate study used an adenoviral vector encoding a cyto
sine base editor and a gRNA targeting the Angptl3 glutamate-135 codon
(CAA), resulting in ≈35% whole-liver Angptl3 editing and reductions of
blood ANGPTL3 protein levels of ≈50% and of blood cholesterol levels
of ≈20% in wild-type mice.64
The therapeutic effects were accentuated
in LDL receptor knockout mice (which phenocopy the severest form of
inherited hypercholesterolaemia, homozygous familial hypercholester
olaemia), with reductions of blood cholesterol levels of ≈50% and of
blood triglyceride levels of .50%. A study of epigenome editing
co-administered to mice two AAV vectors encoding catalytically dead
SaCas9-KRAB and a gRNA targeting a sequence in the mouse Pcsk9 pro
moter, respectively, resulting in reductions of whole-liver Pcsk9 gene ex
pression of ≈50% and of blood PCSK9 protein levels of 80%, with
concordant reduction of blood cholesterol levels.65
The therapeutic ef
fects appeared to attenuate over the course of 6 months, suggesting that
expression of the Cas protein was waning over time. A study of prime
editing used two AAV vectors encoding a split-intein prime editor to
achieve the insertion of a TGA stop codon into Pcsk9 exon 1, albeit at
low efficiency, ≈13% whole-liver editing.66
Recently, two studies addressed the efficacy and safety of in vivo somatic
editing in non-human primates, specifically cynomolgus monkeys.67,68
Both
studies used adenine base editors in combination with the same gRNA tar
getingthesplicedonorattheendofPCSK9exon1,withthegoalofknocking
outthegenebyincorporatingsomeorallofintron1inthemessengerRNA.
ThegRNAmatchesboththemonkeyandhumanPCSK9sequences;fortuit
ously, readthrough into human PCSK9 intron 1 leads to a ribosome quickly
encountering a stop codon, resulting in the production of a truncated pro
tein spanning the codingsequence of exon1and justthree additionalamino
acids.Ofnote,thegRNA-targetedhumanPCSK9sequencehasminimalnat
urallyoccurringgeneticvariation(conservedin.99.9%ofcataloguedalleles
across populations), which is advantageous for the development of an edit
ingtherapeutic.EachstudyadministeredasingleLNPformulationwithboth
an adenine base editor messenger RNA and the gRNA into monkeys via
intravenous infusion. In one study, the therapeutic effects were relatively
modest, with 26% whole-liver PCSK9 editing and reductions of blood
PCSK9 levelsof 32% and of blood LDL cholesterol levelsof 14% at1 month
aftertreatment.67
Intheother study,thetherapeutic effectsweremuch lar
ger and more compatible with clinical translation, with 66% whole-liver
PCSK9 editing and reductions of blood PCSK9 levels of ≈90% and of blood
LDL cholesterol levels of ≈60% persisting for more than 8 months in an on
going study.68
The predominant editing outcome was the desired A-to-G
single-nucleotide change, with low levels of indel mutations.
With respect to safety, the monkeys in the two studies tolerated the
LNP treatment well, without any adverse clinical events. The lipid and
mRNA components of the treatment were found to be cleared within
2 weeks. Although there were immediate, transient rises in blood trans
aminase levels after treatment, these changes resolved in 1–2 weeks, and
there was no subsequent transaminitis to suggest a cytotoxic immune
response. In the first study, some animals received multiple infusions
of LNPs and developed antibodies against the adenine base editor,
though whether this phenomenon would affect the efficacy of future
LNP treatments is unclear. Both studies assessed for off-target editing,
with the first study using CIRCLE-seq and the second study using
ONE-seq (among other methods) to identify candidate sites.
Although there was low-level off-target editing at one candidate site in
monkey liver (at a site not conserved in the human genome), there
was no evidence of off-target editing in cultured human hepatocytes, in
cluding gRNA-independent editing by the deaminase domain of the base
editor. In the light of these preclinical studies, PCSK9 somatic editing ap
pears to be poised to enter clinical trials in the near future.
4.2 Transthyretin amyloidosis
As recounted in the opening clinical vignette, TTR somatic editing for the
treatment of transthyretin amyloidosis has already met early success in a
clinical trial. Similar to PCSK9 and ANGPTL3, the TTR protein is largely
expressed in the hepatocytes and secreted into the bloodstream. TTR
normally forms tetramers that function as thyroxine and vitamin A trans
porters; destabilized TTR monomers have the potential to form abnormal
aggregates that accumulate and damage the heart (cardiomyopathy) or
nerves (polyneuropathy) or both, causing disease with a high degree of
morbidity and mortality. The aggregates are more likely to form from mu
tant protein, driving the disease process at an earlier age, but can also form
from wild-type protein, causing disease late in life. Accordingly, knockdown
of TTR expression in hepatocytes can address disease caused by either mu
tant or wild-type protein. A short-interfering RNA and an antisense oligo
nucleotide, each targeting the TTR messenger RNA and reducing blood
TTR protein levels by ≈80%, have been demonstrated in trials to confer
clinical benefit and have been approved for medical use.69,70
Preclinical studies in mice and non-human primates administered
LNPs with SpCas9 messenger RNA and a gRNA targeting the Ttr or
TTR gene, in each species achieving up to ≈70% whole-liver editing—
producing the expected indel mutations via NHEJ—and .95% reduc
tions of blood TTR protein levels that persisted for a year in long-term
studies.71,72
In an ongoing clinical trial, one group of three patients re
ceived a very low dose (0.1 mg/kg) of LNPs with SpCas9 messenger
RNA and a gRNA targeting exon 2 of the human TTR gene, and a second
group of three patients received a low dose (0.3 mg/kg) of the LNPs.72
One month after treatment, the first and second groups of patients ex
perienced mean 52 and 87% reductions of TTR levels, respectively, with
one of the patients in the second group having a remarkable 96% reduc
tion—outstripping the therapeutic effects of the short-interfering RNA
and antisense oligonucleotide therapies. None of the patients had trans
aminitis or any serious adverse events from the treatment. The durabil
ity of the patients’ TTR reductions over time, as well as the effects of
higher LNP doses that are being tested in additional patients in the trial,
remain to be seen. Nonetheless, this trial stands out as the first success
ful demonstration of somatic in vivo editing of any kind in human patients.
4.3 Duchenne muscular dystrophy
Besides transthyretin amyloidosis and hypercholesterolaemia, the car
diovascular disease for which a somatic editing therapy appears to be
closest to the clinic is Duchenne muscular dystrophy (DMD); although
its hallmark is dysfunction of the skeletal muscles, a primary cause of
death is cardiac failure. Severe DMD arises from any a plethora of
exon deletion, frameshift, nonsense, or splice-site mutations in the
DMD gene on the X chromosome, with the majority of the mutations
residing in a hotspot spanning from exons 45 to 53 and disrupting the
protein product, dystrophin. Dystrophin is a structural protein that is
part of a complex that strengthens myofibres by linking cytoskeletal pro
teins to the cell membrane and extracellular matrix. Notably, dystrophin
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
12. 90 K. Musunuru
retains much of its function even if several of its middle exons are miss
ing, as long as the entire coding sequence is in the frame. This property
allows for therapeutic approaches in which one or more exons are
skipped in order to compensate for any of the aforementioned DMD
mutations, rather than direct correction of the pathogenic mutation.
One such approach is to use standard nuclease gene editing with two
gRNAs to effect NHEJ-mediated deletion of an exon or exons, and an
other approach is to use standard nuclease gene editing to disrupt a
splice site or splicing regulatory site to force the exclusion of an exon
from the Dmd transcript. These approaches are mutation-agnostic
and thus can be applied to entire subsets of DMD patients.
Unlike the liver, the only effective and clinically relevant approach
available for systemic delivery of an editing tool into the skeletal muscles
and heart in vivo is the use of AAV vectors. Four early proof-of-concept
preclinical studies of CRISPR editing to treat DMD used the same mouse
model, the mdx mouse, which has a nonsense mutation in exon 23 of the
Dmd gene. Two of the studies employed intravenous, systemic adminis
tration of dual AAVs in which one vector expressed SaCas9 and the
other vector expressed two gRNAs targeting sites flanking exon
23.73,74
Another study systemically administered a single AAV vector
with SaCas9 and two gRNAs to engineer deletion of exons 21, 22,
and 23.75
The other study used dual AAVs in which one vector ex
pressed SpCas9 and the other vector expressed a gRNA that targeted
the mutant sequence in exon 23 and a gRNA targeted towards the 3′
end of the exon, preventing the exon’s inclusion in the Dmd transcript.76
All four of these studies demonstrated low levels of editing that none
theless led to substantial enough restoration of dystrophin expression
in the skeletal muscles and heart to achieve significant improvements
in muscle function.
In a long-term mouse study that assessed mdx mice following systemic
treatment with dual AAVs expressing SaCas9 and two gRNAs targeting
sites flanking exon 23, Dmd editing and dystrophin expression persisted
in the skeletal muscles and heart for a year.77
Notwithstanding these en
couraging results, treated adult mice but not neonatal mice developed
antibodies as well as T-cell responses against SaCas9. Notably, an un
biased assessment of editing outcomes at the Dmd target site found
that integration of AAV vector sequences was the most frequent
event—an observation mirroring those of previous studies in which
AAV sequences were observed to efficiently incorporate into the sites
of DSBs—raising the possibility of unexpected genotoxic effects, though
no adverse consequences were noted during the course of this study.
The potential liabilities of foreign DNA integration inherent in the use
of AAV vectors in combination with standard nuclease gene editing
can be mitigated through the use of CRISPR tools that do not induce
DSBs. Adenine base editing has proven to be an effective alternative
to standard nuclease gene editing for the treatment of DMD in mouse
models. In a mouse model with a deletion of exon 51 of the Dmd
gene, a split-intein strategy was used to deliver an adenine base editor
via two AAV vectors, targeting and disrupting the splice donor site of
exon 50.78
The editing resulted in the skipping of exon 50, bringing
the in-frame combination of exon 49 and exon 52 together and rescuing
protein function. When administered in vivo via intramuscular injection,
the treatment partially restored dystrophin expression in myofibres. In
another study, an adenine base editor delivered intramuscularly using a
dual-AAV trans-splicing approach was able to directly correct a non
sense mutation in exon 20 of the Dmd gene at a low frequency and mod
estly restore dystrophin expression—a notable result, since it points to
the possibility of treating genetic diseases in which single-nucleotide mu
tations cannot be mitigated through exon-skipping approaches.79
Three studies have progressed beyond mouse models to demon
strate proof of concept of somatic editing treatments for DMD in large
animal models. In a dog model of DMD harbouring a splice site mutation
that results in the exclusion of exon 50, intravenous administration of an
AAV vector expressing SpCas9 and an AAV vector expressing a single
gRNA targeting a site near the 5′
end of exon 51 induced both small
frameshift mutations—many of which restored the reading frame of
the transcript—and deletions affecting an exonic splicing enhancer—
which resulted in skipping of exon 51 in addition to exon 50 and thereby
restored the reading frame.80
The editing resulted in partial restoration
of dystrophin expression in the heart and skeletal muscles throughout
the body. In a pig model of DMD lacking exon 52, administration of
dual AAV vectors expressing split-intein SpCas9 and two gRNAs target
ing sites flanking exon 51, thereby deleting exon 51 and restoring the
reading frame, partially restored dystrophin expression in the muscles
and improved skeletal muscle function and mobility.81
The treatment
also increased the lifespan of the pigs by reducing cardiac arrhythmo
genic vulnerability and staving off the sudden cardiac death that was
the primary cause of mortality in this DMD model. Balancing the posi
tive results of these studies is a more recent study in DMD dog models
that demonstrated that the therapeutic effect of AAV-SpCas9 in re
storing dystrophin expression was accompanied by substantial
SpCas9-specific humoral and cytotoxic T-lymphocyte responses that
resulted in muscle inflammation and in attenuation of dystrophin ex
pression over time, despite the use of immunosuppression.82
The use of AAV vectors to systemically deliver editing tools to the
skeletal and cardiac muscles in vivo in human patients will entail overcom
ing substantial challenges, such as pre-existing immunity preventing
AAV treatment,83
the induction of immune responses that limit the ef
fectiveness of treatment and prevent re-treatment,82
and the current
need for large AAV doses that can induce life-threatening liver toxici
ties. Further development of AAV technologies—such as the directed
evolution of novel muscle-tropic AAV serotypes that avoid the liver
and allow for substantially lower dosing84
—and methods to mitigate
any treatment-associated immune responses will be needed to bring
safe and effective DMD editing therapies within reach.
4.4 Additional considerations for
therapeutic applications
Although therapeutic applications of CRISPR technologies appear to
have much promise, commentators frequently raise two concerns.
First, potential genotoxicity is a major barrier to the deployment of
CRISPR editing therapies. This remains a purely theoretical concern,
as there have been no reports of unintended adverse clinical conse
quences specifically from off-target editing in any treated animal models
or in any of the patients who have received somatic editing therapies in
clinical trials to date. As such, regulatory agencies must balance the the
oretical risks of editing against the projected benefits of editing therapies
—with relatively more to be gained for grievous genetic disorders with
substantial unmet need, and relatively less to be gained for milder dis
eases for which there are already approved therapies—in deciding
whether to allow clinical trials to commence. Second, there could be po
tential downsides to ‘one-and-done’ therapies: what is beneficial at the
time of treatment might become detrimental at a later time in life (e.g. an
antihypertensive therapy that permanently reduces blood pressure), and
there might be moral hazard incurred by treatment (e.g. a therapy to
permanently reduce LDL cholesterol levels and future risk of coronary
heart disease could encourage some patients to thereafter make
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
13. CRISPR and cardiovascular diseases 91
unhealthy lifestyle choices). This concern relies on the assumption that it
will be impossible to undo the effects of an editing therapy, when in fact
several editing modalities—base editing, prime editing, and epigenome
editing—lend themselves to direct, precise reversal in the future should
the need arise.
5. Conclusion
CRISPR editing unequivocally has transformed biomedical research and
promises to do the same for cardiovascular medicine, ushering in a new
paradigm of ‘one-and-done’ therapies to tackle common conditions like
coronary heart disease and rare genetic disorders alike. The prolifer
ation of CRISPR technologies in the decade since the first demonstra
tions of CRISPR-based mammalian gene editing in 2012 promises even
more explosive growth to come in the next decade—to the ultimate
benefit of scientists and patients everywhere.
Conflict of interest: K.M. is an advisor to and holds equity in Verve
Therapeutics and Variant Bio.
Data availability
As this is a review article, there are no data to make available.
References
1. Stein R. He inherited a devastating disease. A CRISPR gene-editing breakthrough stopped
it. https://www.npr.org/sections/health-shots/2021/06/26/1009817539/he-inherited-a-
devastating-disease-a-crispr-gene-editing-breakthrough-stopped-it (5 November 2021,
date last accessed).
2. Musunuru K. A brief history and primer on genome editing. In: Musunuru K, ed. Genome
Editing: A Practical Guide to Research and Clinical Applications. 1st ed. Cambridge, MA:
Academic Press; 2021. p1–19.
3. Rouet P, Smih F, Jasin M. Introduction of double-strand breaks into the genome of mouse
cells by expression of a rare-cutting endonuclease. Mol Cell Biol 1994;14:8096–8106.
4. Bibikova M, Golic M, Golic KG, Carroll D. Targeted chromosomal cleavage and mutagen
esis in Drosophila using zinc-finger nucleases. Genetics 2002;161:1169–1175.
5. Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S.
Stimulation of homologous recombination through targeted cleavage by chimeric nu
cleases. Mol Cell Biol 2001;21:289–297.
6. Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR-Cas nucleases, base edi
tors, transposases and prime editors. Nat Biotechnol 2020;38:824–844.
7. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable
dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;
337:816–821.
8. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X,
Makarova KS, Koonin EV, Sharp PA, Zhang F. In vivo genome editing using Staphylococcus
aureus Cas9. Nature 2015;520:186–191.
9. Walton RT, Christie KA, Whittaker MN, Kleinstiver BP. Unconstrained genome target
ing with near-PAMless engineered CRISPR-Cas9 variants. Science 2020;368:290–296.
10. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P,
Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cpf1 is a single
RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 2015;163:759–771.
11. Strecker J, Jones S, Koopal B, Schmid-Burgk J, Zetsche B, Gao L, Makarova KS, Koonin EV,
Zhang F. Engineering of CRISPR-Cas12b for human genome editing. Nat Commun 2019;
10:212.
12. Liu JJ, Orlova N, Oakes BL, Ma E, Spinner HB, Baney KLM, Chuck J, Tan D, Knott GJ,
Harrington LB, Al-Shayeb B, Wagner A, Brötzmann J, Staahl BT, Taylor KL, Desmarais
J, Nogales E, Doudna JA. CasX enzymes comprise a distinct family of RNA-guided gen
ome editors. Nature 2019:566:218–223.
13. Leibowitz ML, Papathanasiou S, Doerfler PA, Blaine LJ, Sun L, Yao Y, Zhang CZ, Weiss
MJ, Pellman D. Chromothripsis as an on-target consequence of CRISPR-Cas9 genome
editing. Nat Genet 2021;53:895–905.
14. Musunuru K. Assessing for off-target mutagenesis. In: Musunuru K, ed. Genome Editing: A
Practical Guide to Research and Clinical Applications. 1st ed. Cambridge, MA: Academic
Press; 2021. p81–100.
15. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK.
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
Nature 2016;529:490–495.
16. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9
nucleases with improved specificity. Science 2016;351:84–88.
17. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease speci
ficity using truncated guide RNAs. Nat Biotechnol 2014;32:279–284.
18. Yin H, Song CQ, Suresh S, Kwan SY, Wu Q, Walsh S, Ding J, Bogorad RL, Zhu LJ, Wolfe
SA, Koteliansky V, Xue W, Langer R, Anderson DG. Partial DNA-guided Cas9 enables
genome editing with reduced off-target activity. Nat Chem Biol 2018;14:311–316.
19. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base
in genomic DNA without double-stranded DNA cleavage. Nature 2016;533:420–424.
20. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR.
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.
Nature 2017;551:464–471.
21. Koblan LW, Arbab M, Shen MW, Hussmann JA, Anzalone AV, Doman JL, Newby GA,
Yang D, Mok B, Replogle JM, Xu A, Sisley TA, Weissman JS, Adamson B, Liu DR.
Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library ana
lysis, and machine learning. Nat Biotechnol 2021;39:1414–1425.
22. Zuo E, Sun Y, Wei W, Yuan T, Ying W, Sun H, Yuan L, Steinmetz LM, Li Y, Yang H.
Cytosine base editor generates substantial off-target single-nucleotide variants in mouse
embryos. Science 2019;364:289–292.
23. Jin S, Zong Y, Gao Q, Zhu Z, Wang Y, Qin P, Liang C, Wang D, Qiu JL, Zhang F, Gao C.
Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice.
Science 2019;364:292–295.
24. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C,
Newby GA, Raguram A, Liu DR. Search-and-replace genome editing without double-
strand breaks or donor DNA. Nature 2019; 576:149–157.
25. Nelson JW, Randolph PB, Shen SP, Everette KA, Chen PJ, Anzalone AV, An M, Newby
GA, Chen JC, Hsu A, Liu DR. Engineered pegRNAs improve prime editing efficiency.
Nat Biotechnol 2022;40:402–410.
26. Chen PJ, Hussmann JA, Yan J, Knipping F, Ravisankar P, Chen PF, Chen C, Nelson JW,
Newby GA, Sahin M, Osborn MJ, Weissman JS, Adamson B, Liu DR. Enhanced prime
editing systems by manipulating cellular determinants of editing outcomes. Cell 2021;
184:5635–5652.e29.
27. Choi J, Chen W, Suiter CC, Lee C, Chardon FM, Yang W, Leith A, Daza RM, Martin B,
Shendure J. Precise genomic deletions using paired prime editing. Nat Biotechnol 2022;40:
218–226.
28. Jiang T, Zhang XO, Weng Z, Xue W. Deletion and replacement of long genomic se
quences using prime editing. Nat Biotechnol 2022;40:227–234.
29. Anzalone AV, Gao XD, Podracky CJ, Nelson AT, Koblan LW, Raguram A, Levy JM,
Mercer JAM, Liu DR. Programmable deletion, replacement, integration and inversion
of large DNA sequences with twin prime editing. Nat Biotechnol 2022;40:731–740.
30. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing
CRISPR as an RNA-guided platform for sequence-specific control of gene expression.
Cell 2013;152:1173–1183.
31. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N,
Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS.
CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell
2013;154:442–451.
32. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C,
Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman JS. Genome-scale
CRISPR-mediated control of gene repression and activation. Cell 2014;159:647–661.
33. Nuñez JK, Chen J, Pommier GC, Cogan JZ, Replogle JM, Adriaens C, Ramadoss GN, Shi
Q, Hung KL, Samelson AJ, Pogson AN, Kim JYS, Chung A, Leonetti MD, Chang HY,
Kampmann M, Bernstein BE, Hovestadt V, Gilbert LA, Weissman JS. Genome-wide pro
grammable transcriptional memory by CRISPR-based epigenome editing. Cell 2021;202
(184):2503–2519.e17.
34. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step
generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated gen
ome engineering. Cell 2013;153:910–918.
35. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice
carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.
Cell 2013; 154:1370–1379.
36. Gurumurthy CB, O’Brien AR, Quadros RM, Adams J Jr, Alcaide P, Ayabe S, Ballard J, Batra
SK, Beauchamp MC, Becker KA, Bernas G, Brough D, Carrillo-Salinas F, Chan W, Chen
H, Dawson R, DeMambro V, D’Hont J, Dibb KM, Eudy JD, Gan L, Gao J, Gonzales A,
Guntur AR, Guo H, Harms DW, Harrington A, Hentges KE, Humphreys N, Imai S,
Ishii H, Iwama M, Jonasch E, Karolak M, Keavney B, Khin NC, Konno M, Kotani Y,
Kunihiro Y, Lakshmanan I, Larochelle C, Lawrence CB, Li L, Lindner V, Liu XD,
Lopez-Castejon G, Loudon A, Lowe J, Jerome-Majewska LA, Matsusaka T, Miura H,
Miyasaka Y, Morpurgo B, Motyl K, Nabeshima YI, Nakade K, Nakashiba T, Nakashima
K, Obata Y, Ogiwara S, Ouellet M, Oxburgh L, Piltz S, Pinz I, Ponnusamy MP, Ray D,
Redder RJ, Rosen CJ, Ross N, Ruhe MT, Ryzhova L, Salvador AM, Alam SS, Sedlacek R,
Sharma K, Smith C, Staes K, Starrs L, Sugiyama F, Takahashi S, Tanaka T, Trafford
AW, Uno Y, Vanhoutte L, Vanrockeghem F, Willis BJ, Wright CS, Yamauchi Y, Yi X,
Yoshimi K, Zhang X, Zhang Y, Ohtsuka M, Das S, Garry DJ, Hochepied T, Thomas P,
Parker-Thornburg J, Adamson AD, Yoshiki A, Schmouth JF, Golovko A, Thompson
WR, Lloyd KCK, Wood JA, Cowan M, Mashimo T, Mizuno S, Zhu H, Kasparek P,
Liaw L, Miano JM, Burgio G. Reproducibility of CRISPR-Cas9 methods for generation
of conditional mouse alleles: a multi-center evaluation. Genome Biol 2019;20:171.
37. Liu Y, Du Y, Xie W, Zhang F, Forrest D, Liu C. Generation of conditional knockout mice
by sequential insertion of two loxP sites in cis using CRISPR/Cas9 and single-stranded
DNA oligonucleotides. Methods Mol Biol 2019; 1874:191–210.
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
14. 92 K. Musunuru
38. Yu HH, Zhao H, Qing YB, Pan WR, Jia BY, Zhao HY, Huang XX, Wei HJ. Porcine zygote
injection with Cas9/sgRNA results in DMD-modified pig with muscle dystrophy. Int J Mol
Sci 2016;17:1668.
39. Gabriel GC, Devine W, Redel BK, Whitworth KM, Samuel M, Spate LD, Cecil RF, Prather
RS, Wu Y, Wells KD, Lo CW. Cardiovascular development and congenital heart disease
modeling in the pig. J Am Heart Assoc 2021;10:e021631.
40. Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, Wagers AJ, Church
GM. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods 2016;13:
868–874.
41. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O,
Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ,
Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F.
CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 2014;159:
440–455.
42. Yin H, Song CQ, Suresh S, Wu Q, Walsh S, Rhym LH, Mintzer E, Bolukbasi MF, Zhu LJ,
Kauffman K, Mou H, Oberholzer A, Ding J, Kwan SY, Bogorad RL, Zatsepin T, Koteliansky
V, Wolfe SA, Xue W, Langer R, Anderson DG. Structure-guided chemical modification
of guide RNA enables potent non-viral in vivo genome editing. Nat Biotechnol 2017;35:
1179–1187.
43. Banskota S, Raguram A, Suh S, Du SW, Davis JR, Choi EH, Wang X, Nielsen SC, Newby
GA, Randolph PB, Osborn MJ, Musunuru K, Palczewski K, Liu DR. Engineered virus-like
particles for efficient in vivo delivery of therapeutic proteins. Cell;2022;185:250–265.e16.
44. Carroll KJ, Makarewich CA, McAnally J, Anderson DM, Zentilin L, Liu N, Giacca M,
Bassel-Duby R, Olson EN. A mouse model for adult cardiac-specific gene deletion
with CRISPR/Cas9. Proc Natl Acad Sci USA 2016;113:338–343.
45. Guo Y, VanDusen NJ, Zhang L, Gu W, Sethi I, Guatimosim S, Ma Q, Jardin BD, Ai Y,
Zhang D, Chen B, Guo A, Yuan GC, Song LS, Pu WT. Analysis of cardiac myocyte mat
uration using CASAAV, a platform for rapid dissection of cardiac myocyte gene function
in vivo. Circ Res 2017;120:1874–1888.
46. Musunuru K, Sheikh F, Gupta RM, Houser SR, Maher KO, Milan DJ, Terzic A, Wu JC,
American Heart Association Council on Functional Genomics and Translational
Biology; Council on Cardiovascular Disease in the Young; and Council on
Cardiovascular and Stroke Nursing. Induced pluripotent stem cells for cardiovascular dis
ease modeling and precision medicine: a scientific statement from the American heart
association. Circ Genom Precis Med 2018;11:e000043.
47. Ma N, Zhang JZ, Itzhaki I, Zhang SL, Chen H, Haddad F, Kitani T, Wilson KD, Tian L,
Shrestha R, Wu H, Lam CK, Sayed N, Wu JC. Determining the pathogenicity of a gen
omic variant of uncertain significance using CRISPR/Cas9 and human-induced pluripotent
stem cells. Circulation 2018;138:2666–2681.
48. Lv W, Qiao L, Petrenko N, Li W, Owens AT, McDermott-Roe C, Musunuru K.
Functional annotation of TNNT2 variants of uncertain significance with genome-edited
cardiomyocytes. Circulation 2018;138:2852–2854.
49. Wang X, Musunuru K. Confirmation of causal rs9349379-PHACTR1 expression quan
titative trait locus in human-induced pluripotent stem cell endothelial cells. Circ Genom
Precis Med 2018;11:e002327.
50. Doench JG. Am I ready for CRISPR? A user’s guide to genetic screens. Nat Rev Genet
2018;19:67–80.
51. Emmer BT, Hesketh GG, Kotnik E, Tang VT, Lascuna PJ, Xiang J, Gingras AC, Chen XW,
Ginsburg D. The cargo receptor SURF4 promotes the efficient cellular secretion of
PCSK9. Elife 2018;7:e38839.
52. Emmer BT, Sherman EJ, Lascuna PJ, Graham SE, Willer CJ, Ginsburg D. Genome-scale
CRISPR screening for modifiers of cellular LDL uptake. PLoS Genet 2021;17:e1009285.
53. Lucero D, Dikilitas O, Mendelson MM, Islam P, Neufeld EB, Bansal AT, Freeman LA,
Vaisman B, Tang J, Combs CA, Li Y, Voros S, Kullo IJ, Remaley AT. Transgelin: a new
gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen. J
Lipid Res 2021;63:100160.
54. Smith GA, Padmanabhan A, Lau BH, Pampana A, Li L, Lee YC, Pelonero A, Nishino T,
Sadagopan N, Jain R, Natarajan P, Wu RS, Black BL, Srivastava D, Shokat KM, Chorba
JS. CSDE1 is a post-transcriptional regulator of the LDL receptor. bioRxiv 2021,
https://doi.org/10.1101/2020.08.03.235028.
55. VanDusen NJ, Lee JY, Gu W, Butler CE, Sethi I, Zheng Y, King JS, Zhou P, Suo S, Guo Y,
Ma Q, Yuan GC, Pu WT. Massively parallel in vivo CRISPR screening identifies RNF20/40
as epigenetic regulators of cardiomyocyte maturation. Nat Commun 2021;12:4442.
56. Musunuru K, Kathiresan S. Genetics of common, complex coronary artery disease. Cell
2019;177:132–145.
57. Ding Q, Strong A, Patel KM, Ng SL, Gosis BS, Regan SN, Cowan CA, Rader DJ, Musunuru
K. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res
2014;115:488–492.
58. Akcakaya P, Bobbin ML, Guo JA, Malagon-Lopez J, Clement K, Garcia SP, Fellows MD,
Porritt MJ, Firth MA, Carreras A, Baccega T, Seeliger F, Bjursell M, Tsai SQ, Nguyen
NT, Nitsch R, Mayr LM, Pinello L, Bohlooly-Y M, Aryee MJ, Maresca M, Joung JK. In
vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature
2018;561:416–419.
59. Wang X, Raghavan A, Chen T, Qiao L, Zhang Y, Ding Q, Musunuru K. CRISPR-Cas9 target
ing of PCSK9 in human hepatocytes in vivo. Arterioscler Thromb Vasc Biol 2016;36:783–786.
60. Petri K, Kim DY, Sasaki KE, Canver MC, Wang X, Shah H, Lee H, Horng JE, Clement K, Iyer S,
GarciaSP,Guo JA, Newby GA,Pinello L,LiuDR, AryeeMJ, Musunuru K, JoungJK,Pattanayak
V. Global-scale CRISPR gene editor specificity profiling by ONE-seq identifies population-
specific, variant off-target effects. bioRxiv, https://doi.org/10.1101/2021.04.05.438458.
61. Jiang C, Mei M, Li B, Zhu X, Zu W, Tian Y, Wang Q, Guo Y, Dong Y, Tan X. A non-viral
CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo.
Cell Res 2017;27:440–443.
62. Qiu M, Glass Z, Chen J, Haas M, Jin X, Zhao X, Rui X, Ye Z, Li Y, Zhang F, Xu Q. Lipid
nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-
specific in vivo genome editing of Angptl3. Proc Natl Acad Sci USA 2021;118:
e2020401118.
63. Chadwick AC, Wang X, Musunuru K. In vivo base editing of PCSK9 (proprotein conver
tase subtilisin/kexin type 9) as a therapeutic alternative to genome editing. Arterioscler
Thromb Vasc Biol 2017;37:1741–1747.
64. Chadwick AC, Evitt NH, Lv W, Musunuru K. Reduced blood lipid levels with in vivo
CRISPR-Cas9 base editing of ANGPTL3. Circulation 2018;137:975–977.
65. Thakore PI, Kwon JB, Nelson CE, Rouse DC, Gemberling MP, Oliver ML, Gersbach CA.
RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors. Nat
Commun 2018;9:1674.
66. Zheng C, Liang SQ, Liu B, Liu P, Kwan SY, Wolfe SA, Xue W. A flexible split prime editor
using truncated reverse transcriptase improves dual AAV delivery in mouse liver. Mol
Ther 2022;30:1343–1351.
67. Rothgangl T, Dennis MK, Lin PJC, Oka R, Witzigmann D, Villiger L, Qi W, Hruzova M,
Kissling L, Lenggenhager D, Borrelli C, Egli S, Frey N, Bakker N, Walker JA II, Kadina
AP, Victorov DV, Pacesa M, Kreutzer S, Kontarakis Z, Moor A, Jinek M, Weissman D,
Stoffel M, van Boxtel R, Holden K, Pardi N, Thöny B, Häberle J, Tam YK, Semple SC,
Schwank G. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol
levels. Nat Biotechnol 2021;39:949–957.
68. Musunuru K, Chadwick AC, Mizoguchi T, Garcia SP, DeNizio JE, Reiss CW, Wang K, Iyer
S, Dutta C, Clendaniel V, Amaonye M, Beach A, Berth K, Biswas S, Braun MC, Chen HM,
Colace TV, Ganey JD, Gangopadhyay SA, Garrity R, Kasiewicz LN, Lavoie J, Madsen JA,
Matsumoto Y, Mazzola AM, Nasrullah YS, Nneji J, Ren H, Sanjeev A, Shay M, Stahley MR,
Fan SHY, Tam YK, Gaudelli NM, Ciaramella G, Stolz LE, Malyala P, Cheng CJ, Rajeev KG,
Rohde E, Bellinger AM, Kathiresan S. In vivo CRISPR base editing of PCSK9 durably low
ers cholesterol in primates. Nature 2021;593:429–434.
69. Adams D, Gonzalez-Duarte A, O’Riordan WD, Yang CC, Ueda M, Kristen AV, Tournev I,
SchmidtHH,CoelhoT,BerkJL,LinKP,VitaG,AttarianS,Planté-BordeneuveV,MezeiMM,
Campistol JM, Buades J, Brannagan TH III, Kim BJ, Oh J, Parman Y, Sekijima Y, Hawkins PN,
Solomon SD, Polydefkis M, Dyck PJ, Gandhi PJ, Goyal S, Chen J, Strahs AL, Nochur SV,
Sweetser MT, Garg PP, Vaishnaw AK, Gollob JA, Suhr OB. Patisiran, an RNAi therapeutic,
for hereditary transthyretin amyloidosis. N Engl J Med 2018;379:11–21.
70. Benson MD, Waddington-Cruz M, Berk JL, Polydefkis M, Dyck PJ, Wang AK,
Planté-Bordeneuve V, Barroso FA, Merlini G, Obici L, Scheinberg M, Brannagan TH III,
Litchy WJ, Whelan C, Drachman BM, Adams D, Heitner SB, Conceição I, Schmidt
HH, Vita G, Campistol JM, Gamez J, Gorevic PD, Gane E, Shah AM, Solomon SD,
Monia BP, Hughes SG, Kwoh TJ, McEvoy BW, Jung SW, Baker BF, Ackermann EJ,
Gertz MA, Coelho T. Inotersen treatment for patients with hereditary transthyretin
amyloidosis. N Engl J Med 2018;379:22–31.
71. Finn JD, Smith AR, Patel MC, Shaw L, Youniss MR, van Heteren J, Dirstine T, Ciullo C,
Lescarbeau R, Seitzer J, Shah RR, Shah A, Ling D, Growe J, Pink M, Rohde E, Wood
KM, Salomon WE, Harrington WF, Dombrowski C, Strapps WR, Chang Y, Morrissey
DV. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and per
sistent in vivo genome editing. Cell Rep 2018;22:2227–2235.
72. Gillmore JD, Gane E, Taubel J, Kao J, Fontana M, Maitland ML, Seitzer J, O’Connell D,
Walsh KR, Wood K, Phillips J, Xu Y, Amaral A, Boyd AP, Cehelsky JE, McKee MD,
Schiermeier A, Harari O, Murphy A, Kyratsous CA, Zambrowicz B, Soltys R, Gutstein
DE, Leonard J, Sepp-Lorenzino L, Lebwohl D. CRISPR-Cas9 in vivo gene editing for trans
thyretin amyloidosis. N Engl J Med 2021;385:493–502.
73. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Rivera RMC, Madhavan S,
Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA. In vivo genome edit
ing improves muscle function in a mouse model of Duchenne muscular dystrophy.
Science 2016;351:403–407.
74. Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu
EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. In vivo
gene editing in dystrophic mouse muscle and muscle stem cells. Science 2016;351:
407–411.
75. El Refaey M, Xu L, Gao Y, Canan BD, Adesanya TMA, Warner SC, Akagi K, Symer DE,
Mohler PJ, Ma J, Janssen PML, Han R. In vivo genome editing restores dystrophin expres
sion and cardiac function in dystrophic mice. Circ Res 2017;121:923–929.
76. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, Bhattacharyya S,
Shelton JM, Bassel-Duby R, Olson EN. Postnatal genome editing partially restores dys
trophin expression in a mouse model of muscular dystrophy. Science 2016;351:400–403.
77. Nelson CE, Wu Y, Gemberling MP, Oliver ML, Waller MA, Bohning JD, Robinson-Hamm
JN, Bulaklak K, Castellanos Rivera RM, Collier JH, Asokan A, Gersbach CA. Long-term
evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat
Med 2019;25:427–432.
78. Chemello F, Chai AC, Li H, Rodriguez-Caycedo C, Sanchez-Ortiz E, Atmanli A,
Mireault AA, Liu N, Bassel-Duby R, Olson EN. Precise correction of Duchenne mus
cular dystrophy exon deletion mutations by base and prime editing. Sci Adv 2021;7:
eabg4910.
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023
15. CRISPR and cardiovascular diseases 93
79. Ryu SM, Koo T, Kim K, Lim K, Baek G, Kim ST, Kim HS, Kim DE, Lee H, Chung E, Kim JS.
Adenine base editing in mouse embryos and an adult mouse model of Duchenne mus
cular dystrophy. Nat Biotechnol 2018;36:536–539.
80. Amoasii L, Hildyard JCW, Li H, Sanchez-Ortiz E, Mireault A, Caballero D, Harron R,
Stathopoulou TR, Massey C, Shelton JM, Bassel-Duby R, Piercy RJ, Olson EN. Gene edit
ing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.
Science 2018;362:86–91.
81. Moretti A, Fonteyne L, Giesert F, Hoppmann P, Meier AB, Bozoglu T, Baehr A, Schneider
CM, Sinnecker D, Klett K, Fröhlich T, Rahman FA, Haufe T, Sun S, Jurisch V, Kessler B,
Hinkel R, Dirschinger R, Martens E, Jilek C, Graf A, Krebs S, Santamaria G, Kurome M,
Zakhartchenko V, Campbell B, Voelse K, Wolf A, Ziegler T, Reichert S, Lee S,
Flenkenthaler F, Dorn T, Jeremias I, Blum H, Dendorfer A, Schnieke A, Krause S,
Walter MC, Klymiuk N, Laugwitz KL, Wolf E, Wurst W, Kupatt C. Somatic gene editing
ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne
muscular dystrophy. Nat Med 2020;26:207–214.
82. Hakim CH, Kumar SRP, Pérez-López DO, Wasala NB, Zhang D, Yue Y, Teixeira J, Pan X,
Zhang K, Million ED, Nelson CE, Metzger S, Han J, Louderman JA, Schmidt F, Feng F,
Grimm D, Smith BF, Yao G, Yang NN, Gersbach CA, Chen SJ, Herzog RW, Duan D.
Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy
in multiple dystrophic canine models. Nat Commun 2021; 12:6769.
83. Charlesworth CT, Deshpande PS, Dever DP, Camarena J, Lemgart VT, Cromer MK,
Vakulskas CA, Collingwood MA, Zhang L, Bode NM, Behlke MA, Dejene B, Cieniewicz
B, Romano R, Lesch BJ, Gomez-Ospina N, Mantri S, Pavel-Dinu M, Weinberg KI,
Porteus MH. Identification of preexisting adaptive immunity to Cas9 proteins in humans.
Nat Med 2019;25:249–254.
84. Tabebordbar M, Lagerborg KA, Stanton A, King EM, Ye S, Tellez L, Krunnfusz A, Tavakoli
S, Widrick JJ, Messemer KA, Troiano EC, Moghadaszadeh B, Peacker BL, Leacock KA,
Horwitz N, Beggs AH, Wagers AJ, Sabeti PC. Directed evolution of a family of AAV cap
sid variants enabling potent muscle-directed gene delivery across species. Cell 2021;184:
4919–4938.e22.
Downloaded
from
https://academic.oup.com/cardiovascres/article/119/1/79/6564520
by
guest
on
19
March
2023