a new method in gene editing first discovered in bacteria but nowadays is used in knocking out or modifying gene changes .

1. CRISPR/Cas System
And Its
Applications
Gathered by : Afagh Bapirzadeh
Molecular Genetics Doctorate level Student
Supervised by :Dr.Taheri

2. Everything started here when a virus called bacteriophage
attacks a bacteria and insert its genome to bacteria to infect it .

3. CRISPR /Cas
Discovery
In E.Coli
CRISPR history:discovery,characterization,and prosperity by Wenyuan Han et al 2017

4. What is CRISPR and where does it come from originally?
• In 1987 microbiologists found that Archaea and bacteria have evolved adaptive
immune defenses against foreign nucleic acids from cellular invaders
• CRISPR : Clustered Regularly Interspaced Short Palindromic Repeats
• Cas : CRISPR-associated systems

5. CRISPR system

6. Basic mechanism of CRISPR/Cas system
Interference
Interference occur at DNA level A novel interference activity by 3 parts required in host:cas 3
nuclease /spacer complementary/cas gene cascade
CrRNA Biogenesis
Generating small RNAs from pre-RNA with a single
spacer unit(crRNA)
Transcription of CRISPR loci and producing
precursor RNAs
Adaptation
Exposing to phage again and showing resistance ,confirming
that spacers that match phage genome provide immunity
Host can acquire new spacers from extrachromosomal
genetic element

8. How many CRISPR /Cas system exist:
Classification & Nomenclature
• There are three main mechanisms of CRISPR/Cas system
1. Type I
2. Type II
3. Type III
The classification are based on:
comparative genomic analysis,structures,and biochemical activities.
Their prominent signature proteins are respectively Cas3,Cas9
and Cas 10.
Classification and nomenclature of CRISPR-Cas systems: where from here by Makarova et al 2018

10. PAM =Protospacer Adjacent Motif
• It is a specific sequence in the viral or invading genome that Cas proteins (such as
Cas9)bind .
• Different Cas proteins from different species of bacteria or Archea recognize
different PAM sites.
• The most commonly used PAM is 5´-NGG- 3´ that cas9 in S.pyogenes recognize.

11. Function of System type III
• In type III CRISPR system there are 2
homologous :
• Cmr –mediated DNA interference
• No PAM needed
• DNA/RNA interference activity
• Csm is very similar to Cmr
• In Csm 3 units exist and they cleave
complementary ssRNA with 6 nt intervals .
• There is a self –distinguishing mechanism at
RNA level which the DNA cleavage is inactivated
by target RNA cleavage of CSM 3 or CSM 4
Cmr
complexes
Several
Cmr4+Cmr5
complexes
Cmr2+Cmr3
anchor 5
repeat handle
of crRNA
Cmr6+Cmr1
function as a
cap to close
complex

13. Structure Of System Type I
CRISPR/Cas system Type I consist of
1. 6× 𝑪𝒂𝒔𝟕 + 𝑪𝒔𝒆 = 𝒂𝒕𝒕𝒂𝒄𝒉 𝒕𝒐 𝒔𝒑𝒂𝒄𝒆𝒓 𝒐𝒇 𝒄𝒓𝑹𝑵𝑨
2. Cas 3 nuclease
3. Cse1(the largest subunit in the cascade) = recognition of PAM

14. Type II CRISPR system
1.SgRNA:fusion of crRNA+tracRNA
2.Cas9

15. Type II CRISPR System
CRISPR history:discovery,characterization,and prosperity by Wenyuan Han et al 2017

17. Cas9 Subunits And Their Function
=Recognition lobe
Cleaves complementary
strand to crRNA Cleaves opposite strand

18. NHEJ & HD Repair system in CRISPR
CRISPR-mediated genome editing and human diseases by Liquan cai et al 2016

19. Use of NHEJ and HDR
Gene Knockout
• By introducing indels and frameshifts into the coding regions of a gene
with nonsense-mediated decay of the mRNA transcript knock a gene out.
Gene Deletion
• Paired nucleases excise regions of the coding gene resulting in premature
truncation and knockout of the protein in a manner more generally efficient
than introducing frameshifts
Gene Correction & Gene Addition
• Require an exogenous DNA template that can be introduced as either
single-stranded or double-stranded DNA .
• The DNA template contains homologous sequence arms that flank the
region containing the desired mutation or gene cassette.

21. Systems Differences
System type I
PAM needed
Complex of Cas
proteins
System type II
PAM needed
Only 1 Cas
protein(Cas9)
System type III
NO PAM
Complex of Cas
proteins

22. Factors Affecting Efficacy Of The CRISPR/Cas9 System
Target DNA site selection
sgRNA design
Off-target cutting
Incidence /efficiency of HDR versus NHEJ
Cas9 activity
Method of delivery
Still the major obstacle for use of CRISPR for
in vivo applications

23. Target DNA site selection and sgRNA design
• CRISPR/Cas system is able to target any 23 bp sequence that contains PAM motif on the
either strand of DNA.
• SpCas9 PAM occur every 8 bps
• Directed evolution and structure-guided rational design has allowed for engineering of Cas9
variants with altered PAM sequence specificity
• These are not easy tasks
• Maybe this system is less specific due to shorter targeting sequence
• Transformed DNA are then amplified by PCR and purified by agarose gel electrophoresis
,DNA sequencing-gal assay, Western blotting and co-purification

24. Off-target Cutting
On-target performance means whatever our change is(cutting ,deleting ,adding…)will be happening in
the previously determined place or sequences.
Off-target performance means we have specific location designed or predicted to change but our
changes don’t happen there ,they may happen near our PAM or further away and so this is not a wanted
change .
• Efforts to improve specificity and reduce off-target cutting have resulted in the design of mutant
Cas9 system
In an experiment designed to measure off-
target cleavage of CRISPR/Cas9 system in
human genes, scientists found, in genes
having high sequence homology with our
target genes ,we will have significant off-
target cleavage activities
CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity by Cradick et al 2013

25. Elements Influencing
Off/On-target Activities
 Off-target cleavage activity of CRISPR/Cas9 system is influenced by genomic context
 Mismatches in, or proximal to, the PAM sequence could block cleavage in some of
plasmids
 Transfection conditions ,including plasmid dosage ,may be optimized to decrease off-
target cleavage, although the effects may vary with guide strands.
 Although the ability of engineered CRISPR/Cas9 systems to target multiple sites/genes
with different guide strands is an exciting feature, each system may lead to off-target
cleavage
 CRISPR –directed on and off –target cleavage can induce a wide range of
indels,with a large number of one-base insertions and a few large deletions.
According to investigation on human beta-globin
and CCR5 genes conducted by Cradick and Fine
in 2013:

26. Incidence/Efficiency Of HDR
Incidence of HDR-mediated DNA repair is typically
extremely low in mammalian cells
NHEJ repair is more frequent in the CRISPR/Cas9
editing system-even in the presence of donor
template DNA
Several approaches that emerged to increase HDR
efficiency and suppress NHEJ :
1.Use of small molecular inhibitors of NHEJ
2.Gene silencing
3.Cellcycle synchronization
4.Use of cell lines deficient in NHEJ components

27. Cas9 Activity
• Cas9 proteins which are identified and used for gene editing
 Staphylococcus aureus(SaCas9)
 Neisseria meningitidis(NmCas9)
 S.thermophiles(St1Cas9)
• Each of Cas9 proteins has different PAM sequences and variable activity.
• Factors that influence Cas9 activity :
1. Increasing Cas9 access to NLS (to increase the chance of translocation of Cas9 into the nucleus in
Eukaryotic cells)by adding a 32 amino acid spacer between NLS and Cas9
2. Increasing on-target cutting activity by increasing sgRNA concentration on Cas9 protein
• Undesirable Cas9 features:
1. It is a quite low enzyme with a single turnover rate of 0.3-1 (1/min)
2. It displace from DNA strand even after DSB
3. It is less like a catalytic enzyme and more like a single-shot activator

28. CRISPR delivery
systems
Physical delivery
methods
Viral vector
delivery methods
Non-viral delivery
vehicles
Emerging delivery
technologies
Microinjection
Electroporation
Hydrodynamic
Delivery
Lipid-coated Mesoporous Silica
Particles
Inorganic Nanoparticles
MEND
SLO
1. Adeno-associated
Virus(AAV)
2. Lentivirus
3. Adenovirus
Liposomes
Gold Nano
Particles
DNA
penetratin
g Peptides
Lipoplexes
DNA
Nanoclew
iTop
Delivering CRISPR: a review of the challenges and approaches by A.Lino et al 2018

29. CRISPR/Cas9 system applications

30. Interference CRISPR
I. In order to control gene expression ,scientists
re-engineered a new Cas9 protein called
dCas9,which is catalically-dead Cas9 enzyme
II. dCas9 recognize and bind to a target DNA
sequence and disrupt RNA polymerase or
transcription factor binding
III. So it could repress expression of multiple genes
simultaneously without altering the genome
The CRISPR tool kit for genome editing and beyond by Adil 2018

32. Second Generation CRISPR Gene-editing Tool

33. Alternative To Cas9

36. Gene Editing Application Of CRISPR System
CRISPR/Cas9 genome editing has been used to correct
disease –causing DNA mutations ranging from a single
base pair to large deletions, in model systems ranging
from cells in vitro to animals in vivo.
• The application of the CRISPR/Cas 9 approach only requires
designing the guide RNA (gRNA).
• Multiplex gene alterations are only really possible with use of
CRISPR/Cas9 because multiple gRNAs can be used simultaneously.
• What is always essential in gene editing is a site-specific double
strand breakage in the DNA that both of these main features are
provided when sgRNA binds and then Cas9 cuts .
• CRISPR makes it possible to simultaneously delete multiple genes
.this capacity sets it apart from other gene-editing tools.

37. Genes Labeling Application Of CRISPR System
Blossom of CRISPR technologies and applications in disease treatment by Liu et al 2018

38. CRISPR Epigenetic Functions
Researchers used the programmable capacity of Cas9 to
recruit various epigenetic writers and erasers to a
specific locus
I. A fused protein of dCas9 and acetyltransferase
was developed. It catalyzed acetylation of histone H3
lysine 27 at target sites
II. A fused protein of dCas9 and catalytic domain of
eukaryotic DNA methyl transferase (DNMT3A)
or prokaryotic DNA methyl transferase (MQ3)

39. Hemophilia knock in mice CRISPER in IRAN
‫ب‬‫ه‬‫گزارش‬‫ایران‬ ‫بیوتکنولوژی‬ ‫اطالعات‬ ‫مرکز‬‫از‬ ‫نقل‬ ‫به‬‫ایسنا‬‫حض‬ ‫با‬ ‫مراسمی‬ ‫طی‬ ،‫ور‬
‫روش‬ ‫فناوری‬ ‫محصول‬ ‫اولین‬ ‫از‬ ،‫فناوری‬ ‫و‬ ‫تحقیقات‬ ،‫علوم‬ ‫وزیر‬ ‫غالمی‬ ‫منصور‬ ‫دکتر‬‫نوین‬
‫ژنی‬ ‫ویرایش‬“‫رونمایی‬ ‫کریسپر‬‫شد‬.
‫و‬ ‫ایران‬ ‫در‬ ‫جوان‬ ‫دانشجویان‬ ‫و‬ ‫پژوهشگران‬ ‫از‬ ‫تعدادی‬ ‫علمی‬ ‫حوزه‬ ‫این‬ ‫اهمیت‬ ‫به‬ ‫توجه‬ ‫با‬
‫از‬ ‫فناوری‬ ‫زیست‬ ‫و‬ ‫ژنتیک‬ ‫مهندسی‬ ‫ملی‬ ‫پژوهشگاه‬ ‫ویژه‬‫به‬۳‫مطالعات‬ ‫و‬ ‫فعالیت‬ ‫پیش‬ ‫سال‬
‫جالب‬ ‫نتایج‬ ‫به‬ ‫منجر‬ ‫ها‬‫پژوهش‬ ‫این‬ ‫و‬ ‫کردند‬ ‫آغاز‬ ‫ژن‬ ‫ویرایش‬ ‫متد‬ ‫این‬ ‫روی‬ ‫بر‬ ‫را‬ ‫خود‬
‫شده‬ ‫انسان‬ ‫های‬‫بیماری‬ ‫از‬ ‫بسیاری‬ ‫مدل‬ ‫های‬‫موش‬ ‫تولید‬ ‫جمله‬ ‫از‬ ‫توجهی‬‫است‬.‫در‬
‫کردند‬ ‫استفاده‬ ‫کریسپر‬ ‫ژنوم‬ ‫ویرایش‬ ‫روش‬ ‫از‬ ‫ایرانی‬ ‫محققان‬ ،‫طرح‬ ‫این‬.‫ت‬ ‫این‬ ‫به‬‫که‬ ‫رتیب‬
‫فاکتور‬ ‫ژن‬ ‫ابتدا‬۸‫برای‬ ‫مناسب‬ ‫منطقه‬ ‫و‬ ‫دادند‬ ‫قرار‬ ‫تحلیل‬ ‫و‬ ‫تجزیه‬ ‫مورد‬ ‫را‬ ‫موشی‬
‫کردند‬ ‫انتخاب‬ ‫را‬ ‫گیری‬‫هدف‬.
،‫ادامه‬ ‫در‬RNA‫ورز‬‫دست‬ ‫های‬‫جنین‬ ‫سپس‬ ‫و‬ ‫تزریق‬ ‫موش‬ ‫جنین‬ ‫هسته‬ ‫پیش‬ ‫به‬ ،‫راهنما‬ ‫های‬‫ی‬
‫مولکول‬ ‫های‬‫آزمایش‬ ،‫نوزادان‬ ‫تولد‬ ‫از‬ ‫بعد‬ ‫و‬ ‫شدند‬ ‫منتقل‬ ‫خوانده‬ ‫مادر‬ ‫های‬‫موش‬ ‫رحم‬ ‫به‬ ‫شده‬‫ی‬
‫کرد‬ ‫تأیید‬ ‫را‬ ‫حاصل‬ ‫های‬‫موش‬ ‫در‬ ‫ژن‬ ‫این‬ ‫شدن‬ ‫غیرفعال‬ ،‫بیوشیمیایی‬ ‫آنالیزهای‬ ‫و‬.
‫ب‬ ‫بتوانند‬ ‫امیدوارند‬ ‫پژوهشگاه‬ ‫این‬ ‫محققان‬ ،‫موش‬ ‫حیوانی‬ ‫مدل‬ ‫های‬‫ویژگی‬ ‫به‬ ‫توجه‬ ‫با‬‫این‬ ‫ا‬
‫کنند‬ ‫ایفا‬ ‫انسان‬ ‫های‬‫بیماری‬ ‫درمان‬ ‫و‬ ‫تشخیص‬ ‫به‬ ‫کمک‬ ‫در‬ ‫بسزایی‬ ‫نقش‬ ‫تکنیک‬.

40. Anti-CRISPR System
• In 2013 ,Bondy-Denomy found out that CRISPR/Cas system is not an
invincible system ,through investigating Pseudomonas aeruginosa.
• They observed resistance in phages that were sensitive to CRISPR system.
• 8 genes (anti-CRISPR genes =acr genes)were responsible for phage resistance to CRISPR system
• Acr genes existed in mobile genetic elements(MEGs) that are associated with bacterial CRISPR/Cas
system in II-C Neisseria meningitides.
• Mechanism of these 8 genes function include producing proteins that interact with cascade complex or
Cas3 complex and so elimination of CRISPR immunity either by preventing target DNA binding or the
recruitment of Cas3 to the cascade complex.
• Acr genes are controlled by a putative transcription regulator downstream of all acr genes called anti-
CRISPR associated protein(=Aca)
The use of this new finding in gene editing application is :providing an additional layer
for manipulating CRISPR/Cas system for biotechnological applications

41. Anti-CRISPR System Function

42. Websites to design sgRNA
1. Chopchop CRISPR=http://chopchop.cbu.uib.no/
2. Crispor=http://crispor.tefor.net/
3. E-crispr design=http://www.e-crisp.org/E-CRISP/
4. clontech crispr=www.takarabio.com/
5. Crispr target finder=http://flycrispr.molbio.wisc.edu/tools