A CRISPR/Cas9, works like a biological version of a word-processing programme’s “find and replace”. Its simplicity and extremely low cost of implementation is the reason to use. How Cas 9 is activated and its mechanism (DNA binding and cleavage), it's regulation and application in human disease therapy, new drug screening, agriculture and biofuel etc.

1. CRISPR Cas editing mechanism
Clustered Regularly Interspaced Short Palindromic Repeats
CRIPSR associated protein 9

2. 1987 2002 2005 2006 2008 2010 2012 2013 2014 2016 2017 2019 2020
Nakata and
colleagues
discovered
repeat and non-
repeat
sequences
downstream of
iap gene.
Repeat arrays
were given the
name CRISPR.
Mojica and
colleagues that
“spacers”
contain DNA
from
bacteriophages.
Bolotin et.al
observed the
presence of
endonuclease.
Koonin et al.
proposed that
spacers
produce short
RNA guides.
Upstream of
“protospacers”,
conserved
motifs called
PAM are target
sites for Cas
endonucleases.
Three CRISPR
systems had
been
identified in
bacteria:
Type I, II, III.
Dr. Jennifer
Doudna and Dr.
Emmanauel
Charpentier
published that
CRISPR Cas9 could
be programmed
with RNA to edit
genomic DNA.
The use of
CRISPR began in
StCas and
SpCas9 could be
engineered to
edit mammalian
genomes.
The first
patent of
CRISPR-Cas9,
specific to
plant and
animal cells.
The first application
of CRISPR gene
editing in clinical
treatment.
Development of
base editing
technology.
The CRISPR
Cas9 is used for
the first time to
modify the
beta-globin
gene in human
embryo.
Development
of prime
editing
technology.
A US trial
safely
showed
CRISPR gene
editing on
three cancer
patient.
Timeline of CRISPR

3. Introduction
A CRISPR/Cas9, works like a biological version of a word-processing programme’s “find and replace”.
How the technique works
A cell is transfected with an enzyme
complex containing:
Guide molecule, Healthy DNA copy,
DNA cutting enzyme.
Guide molecule finds the
target DNA strand.
An enzyme cuts off the
target DNA strand.
The defective DNA
strand is replaced with
a healthy copy.
Two important advantages
of CRISPR Cas9 system are:
1) It has remarkable versatility
when it comes to working in
cells as well as directly in the
embryos of multiple species.
2) Its simplicity and extremely low
cost of implementation.
Two essential components:
Guide RNA – to match desire
target gene
Cas9 – endonuclease which break
ds DNA and allow modification to
genome
Figure 1: Image credit: Biorender

4. Figure 2: Cas9 protein. It has six domains: Rec I, Rec II, Bridge Helix, RuvC, HNH and PAM
interacting. Domains are shown in schematic, crystal, and map form.
Cas9
Rec I: the largest and responsible for binding of guide RNA.
Rec II: role is not understood yet.
Bridge Helix: Arginine rich and crucial for initiating cleavage
activity upon binding of target DNA.
PAM interacting: confers PAM specificity and responsible
for initiating for binding to target DNA.
HNH and RuvC: nuclease domains that cut single stranded
DNA.
Guide RNA (gRNA)
Figure 3: Engineered Guide RNA. It is single strand RNA. It forms one tetraloop and and two
or three stem loops.
In engineered CRISPR systems, guide RNA is compromised of
single strand that forms T-shape compromise of one tetra loop
and two or three stem loops.
It have a 5’ end that is complementary to target DNA
sequence.
The Cas9 protein remains “inactive” in absence of the guide RNA.
CRISPR RNA(crRNA) and trans activating CRISPR RNA(tracrRNA)
forms a complex known as gRNA.

5. Transcription
of pre-crRNA
and tracrRNA
Binding of
tracrRNA to
pre-crRNA
Cleavage
of guide
RNA from
pre-crRNA
Formation
of active
Cas9
Transcription
of guide RNA
as a single
sequence
Transcription
and
translation
of Cas9
nuclease
Formation
of active
Cas9
How Cas9 is activated

6. Figure 4: Activation of Cas9 protein by guide RNA binding. Due to conformational change in Cas9
activation of Cas9 nuclease activity.
Figure 5: Target DNA binding and cleavage by Cas9.
Once the Cas9 protein gets activated, it stochastically searches for
target DNA and bind with sequences that matches its PAM sequence.
1) Cas9 scans potential target DNA for the appropriate PAM.
2) When the proteins finds the PAM, the protein-guide RNA complex
will melt the bases immediately upstream of the PAM and pair
them with target complimentary region on the gRNA.
3) If the complimentary region and target region pair properly,
the RuvC and HNH nuclease domains will cut the target DNA after
the third nucleotide base upstream of the PAM.
Cas9 mechanism
Rec I and Rec II bind the
complimentary region of
gRNA.
PAM Interacting and
RuvC bind at stem loops
on gRNA.

7. DNA binding and Cleavage
PAM dependent target DNA binding, melting and recognition by Cas9
1) PAM binding: Arginine residues R1333 and R1335 of PI domain bind to
major groove of Guanine in PAM and lysine residue in Phosphate lock
loop binds to minor groove.
2) Phosphate lock loop: Positions the PAM and target DNA such that
serine 1109 in PLL, and two N of PLL can form H-bonds to phosphate at
position +1 of the PAM.
3) Guide RNA: Target DNA will unzip as the bases flip up and bind gRNA.
The initial PAM binding and stabilization of +1P the gRNA would not be
able to bind to target DNA and Cas9 would be inefficient. It shows high
efficiency and specificity of Cas9.
4) Cleavage: HNH and RuvC cleave between 3 and 4 nucleotides from
PAM. Nucleases cleave individually without affecting ability of Cas9,
which makes CRISPR power fool and flexible genome editing tool.

8. CRISPR methods and techniques
CRISPR gene knock in
CRISPR gene knock out
CRISPRa and CRISPRi
CRISPR screen
Transcription Regulation

9. CRISPR KO/KI
If Cas9 creates a DSB, it will most likely be repaired by
NHEJ. However, NHEJ is error-prone, and it usually results
in insertions and deletions (indels) in the region being
repaired.
When indels occur within the coding region of a gene
and result in a frameshift mutation, the gene becomes
non-functional. This is known as a gene knockout (KO).
In the presence of a DSB induced by Cas9, cells can also
repair themselves via HDR, and this pathway offers an
opportunity for researchers to insert a new piece of DNA
or an entire gene. This method is known as a gene knock-
in.

11. CRISPR Screen
What is CRISPR screening?
CRISPR screening is a large-scale experimental approach used to screen a
population of mutant cells to discover genes involved in a specific phenotype.
How does CRISPR screening works?
The basic idea of CRISPR screening is to knock out every gene that could be
important, although knock out only one gene per cell
Negative and Positive screens
Drug resistance and drug sensitivity are two of the major physiological responses
that are frequently studied by CRISPR screening (Figure 1). Negative screens are
used to find genes that cause drug resistance, and positive screens are used to
find genes that cause drug sensitivity.

13. CRISPRa – CRISPR Activation CRISPRi – CRISPR Interference
CRISPR/Cas9
CRISPRa CRISPRi
dCas9
Still target specific DNA
location
Incapable to cut DNA

14. CRISPR activation or CRISPRa is a variant of CRISPR in which
a catalytically dead (d) Cas9 is fused with a transcriptional
effector to modulate target gene expression.
Once the guide RNA navigates to the genome locus along
with the effector arm, the dCas9 is unable to make a cut,
and instead, the effector activates the downstream gene
expression.
CRISPR interference or CRISPRi is also a variant of
CRISPR in which a catalytically dead (d) Cas9 is fused
with a transcriptional effector to modulate target gene
expression.
However, in CRISPRi, when guide RNA navigates to the genome locus along with the effector arm, it represses the
downstream gene expression instead of activating it.

15. Transcription repression by CRISPRi
The utility of dCas9 for sequence-specific gene repression was first demonstrated
in E. coli as a technology called CRISPR interference (CRISPRi). By pairing dCas9
with a sequence-specific sgRNA, the dCas9–sgRNA complex can interfere with
transcription elongation by blocking RNA polymerase (Pol) .In bacteria, the
CRISPRi method using dCas9 is highly efficient in suppressing genes; is specific,
with minimal off-target effect.
The introduction of CRISPRi into mammalian cells using dCas9 alone achieved
only modest repression of enhanced GFP (egfp) in the human HEK293T reporter
cell line.
When targeting endogenous genes such as the transferrin receptor CD71, C-X-C
chemokine receptor type 4 (CXCR4) and tumour protein 53 (TP53), up to 80%
repression was observed.
To achieve enhanced repression, the Krüppel-associated box (KRAB), was fused
to the carboxyl terminus of dCas9. Together with a target-specific sgRNA, the
dCas9–KRAB fusion proteins can efficiently repress endogenous genes.
. This repression was further enhanced by fusing KRAB to the amino terminus of
dCas9, leading to strong repression of endogenous genes. The level of dCas9- or
KRAB–dCas9-mediated knockdown of endogenous genes was highly dependent
on the sgRNA targeting site, suggesting that the chromatin structure or the
presence of regulatory elements may limit the level of repression.

16. Transcription activation by CRISPRa
CRISPRa, uses dCas9 fusion proteins to recruit transcription activators. A fusion of
dCas9 with the ω-subunit of the E. coli Poll allowed assembly of the holoenzyme at a
target promoter for gene activation in E. Coli.
The fusion of VP64 or of the p65 activation domain (p65AD) to dCas9 in mammalian
cells could activate both reporter genes and endogenous genes, with a single sgRNA.
However, the use of multiple sgRNAs was necessary to achieve significant activation of
the endogenous genes.
sgRNA engineering was also shown to enhance the efficiency of gene activation.
The recruitment of VP64 using protein-interacting RNA aptamers incorporated into the
sgRNA has achieved activation of the gene encoding endogenous zinc-finger protein
using multiple sgRNAs.
synergistic activation mediator (SAM) system, was achieved by adding MS2 aptamers
to the sgRNA; MS2 recruits its cognate MS2 coat protein (MCP) fused to p65AD and
heat shock factor 1.
The SAM technology, together with dCas9–VP64, further increased endogenous gene
activation compared with dCas9–VP64 alone and was shown to activate 10 genes
simultaneously.

17. Base editing & prime editing
Some of the most recently developed CRISPR methods are base editing and prime editing.
Base Editing:- Base editing uses either a catalytically dead Cas9 (dCas9) or a nickase Cas9 (nCas9). dCas9 is
incapable of cutting DNA, while nCas9 produces ‘nicks’, or single-stranded breaks (SSBs) in the DNA.
By fusing either dCas9 or nCas9 to a DNA modifying enzyme, researchers can alter specific nucleotides. One of the
limitations of base editing is that they cannot be used to alter every possible nucleotide.
And this is one of the factors that led to the development of prime editing.
Prime Editing:- Prime editing involves fusing nCas9 to an engineered reverse transcriptase and a prime editing
guide RNA (pegRNA). The pegRNA contains two sections: one that guides to the region of interest, and another
that contains the desired substitution/s for repair after the single-stranded cut has been generated.
After one strand has been altered by the prime editor, the complementary strand can also be corrected - an
additional gRNA and nCas9 will create a nick in the strand and it will be repaired using the previously edited strand
as a template.
Prime editing is predicted to be capable of treating 89% of genetic mutations in humans.

18. Applications

19. Role in Gene therapy
Gene therapy is the process of replacing the defective gene
with exogenous DNA and editing the mutated gene at its
native location.
CRISPR/Cas-9 gene editing has held the promise of curing
most of the known genetic diseases such as sickle cell
disease, β-thalassemia, cystic fibrosis, and muscular
dystrophy.
The genetic mutation of the cystic fibrosis transmembrane
conductance regulator (CFTR) gene decreases the structural
stability and function of CFTR protein leading to cystic
fibrosis.
In 2013, researchers culture intestinal stem cells from two
cystic fibrosis patients and corrected the mutation at the
CFTR locus resulting in the expression of the correct gene
and full function of the protein.

20. Schematic Illustration Showing Functional Repair of CFTR by CRISPR-Cas9 in
Intestinal Stem Cell Organoids Acquired from Patients with Cystic Fibrosis
Reproduced from Schwank et al. 2013.

21. Therapeutic role of CRISPR/Cas9
The first CRISPR-based therapy in the human trial was
conducted to treat patients with refractory lung cancer.
Researchers first extract T-cells from three patient’s blood
and they engineered them in the lab through CRISPR/Cas-9
to delete genes (TRAC, TRBC, and PD-1) that would interfere
to fight cancer cells.
Then, they infused the modified T-cells back into the
patients. The modified T-cells can target specific antigens
and kill cancer cells. Finally, no side effects were observed
and engineered T-cells can be detected up to 9 months of
post-infusion.

22. Drug Discovery
Screening for target sites
The CRISPR library can detect living cells with specific
conditions, such as drug therapy. By using the system,
researchers can identify genes and proteins that cause
or prevent disease, thereby identifying potential drug
targets.
Drug discovery
CRISPR/Cas9 animal models allow scientists to discovery
new drugs more accurately and verify the safety and
efficacy of the drugs, ensuring that these models better
predict what will happen in clinical trials. Up regulating
or down regulating gene activity using the CRISPR/Cas9
system is a subtle way of studying the importance of
genes and proteins that can be activated or inhibited by
drugs to treat disease.

23. Better Biofuel
The major drawbacks of biofuel production at the commercial level are its low yield, non-availability of
feedstock, feedback inhibition, presence of inhibitory pathways in various organisms, and biofuel
intolerance of organisms.
Gene knockout and gene cassette insertions employing CRISPR-
Cas9 in Saccharomyces cerevisiae and Kluyveromyces marxianus
have resulted in enhanced production of bioethanol in these
organisms, respectively.
CRISPR-Cas9 modification of microalgae has demonstrated improved total lipid content, a
prerequisite for biofuel production. All over, CRISPR-Cas9 has emerged as a tool of choice for
engineering the genome and metabolic pathways of organisms for producing industrial biofuel.
In plant-based biofuel production, the biosynthetic pathways of lignin interfere with the
satisfactory release of fermentable sugars thus hampering efficient biofuel production.

24. In Agriculture
Genome editing with CRISPR-Cas9 is amendable to edit
any gene in any plant species. Because of its simplicity,
efficiency, low cost, and the possibility to target multiple
genes, it allows faster genetic modification than other
techniques. It also can be used to genetically modify
plants that were previously neglected.
Impressive genetic modifications have been achieved
with CRISPR-Cas9 to enhance metabolic pathways,
tolerance to biotic (fungal, bacterial or viral pathogens),
or abiotic stresses (cold, drought, salt), improve
nutritional content, increase yield and grain quality,
obtain haploid seeds, herbicide resistance, and others.

25. Generation of an Animal model
One of the most exciting applications of CRISPR-Cas9 is the generation of animal models for the
study of a variety of diseases.
Direct injection of Cas9 mRNA and sgRNA for gene editing of single cell embryos is a new
method for rapid establishment of animal models. This approach has been successfully applied
to the generation of animal models, such as mice, rats, monkeys, zebrafish and cattle.
In particular, transgenic animals can be changed more easily, faster and more efficiently. These
animal models may be important in vivo models for diseases, such as cancer, bone disease,
immunodeficiency disease and many other inherited human diseases.
A good example in the establishment of animal models for tumor
research was done on lung cancer by establishing a Cre-dependent
Cas9 knock in mice.
A prominent example in the study of cardiovascular disease is the
generation of transgenic mice with severe heart failure by using
AAV9 to transfer sgRNA targeting Myh6 locus of cardiomyopathy.
Furthermore, CRISPR-Cas9 system has also been used to establish
animal models of infectious disease like human immunodeficiency
virus (HIV), human papillomaviruses (HPV), and chronic hepatitis B
virus (HBV).

26. References
• https://www.synthego.com/learn/crispr
• https://sites.tufts.edu/crispr/crispr-mechanism
• http://dx.doi.org/10.1098/rstb.2015.0496
• CRISPR Manual: CRISPR Cas9 An introductory Guide for Gene knockout; https://www.abmgood.com
• What is CRISPR/Cas9?; https://www.researchgate.net/publication/301203306
• CRISPR Methods and Protocols: Editors, Magnus Lundgren, Emmanuelle Charpentier, Peter C. Fineran;
http://www.springer.com/series/7651
• https://www.sciencedirect.com/science/article/pii/S2211383521000113#bib37
• https://www.dovepress.com/mechanism-and-applications-of-crisprcas-9-mediated-genome-editing-peer-reviewed-
fulltext-article-BTT

27. Thank you
Presented by: Suchi Patel
Department of Biochemistry and Biotechnology
St. Xavier’s College, Autonomous, Ahmedabad