Anti RISPRs: Protein inhibitor of CRISPR-Cas system Introduction to CRISPR- Cas system

1. 1
By:
CHAHAK JAIN
M.Sc. Biochemistry

2. • CRISPRs (Clustered Regularly
Interspaced short Palindromic
Repeats) is a family of DNA
sequences in bacteria.
• Small cluster of cas ( CRISPR-
associated) gene are located
next to CRISPR Sequences.
• They are essential
components of diverse
adaptive immune systems that
defend bacteria and Achaea
from infection by foreign
genetic elements (phages).
• CRISPR loci consist of multiple
semi-palindromic DNA repeats
of 21 to 48 nucleotides,
interspersed with variable
“spacer” sequences of similar
length.
• The spacers comprise
sequences that are
complementary to mobile
genetic elements, from the
exposure of previous DNA (eg-
phages and plasmids).
2

3. (Seahyoung et al.2015)
3
Cas-CRISPR
Associated Gene
PAM
Protospacer Adjacent Motif

4. • 1987 1st report on CRISPR (Ishano et al.)
• 2000 CRISPR present throughout prokaryotes (Mojica et al.)
• 2005 Foreign elements, proposed immunity function (Mojica et al.)
• 2010 Cas9 is guided by spacer (Garneau et al.)
• 2011 Heterologous expression of CRISPR type II (Sapranauskas et al.)
• 2012 Proposal CRISPR for Genome editing (Jinek, Doudna, Charpentier et al.)
• 2013 CRISPR used for genome editing in eukaryotic cells (Zhang et al.)
• 2014 Crystal structure of Cas9 gRNA complex (Nishimasu, Zhang et al.)
4
Jennifer Daudna Emmanuelle Charpentier Feng Zhang

5. 5

6. 6
CRISPR
CLASS 1
Type1 Type 3 Type 4
CLASS 2
Type 2 Type 5 Type 6
CLASSIFICATION OF CRISPR SYSTEM
 These requires the joint
activity of multiple Cas
proteins to induce the
degradation of foreign DNA or
RNA molecules.
 These involves a single
multi-domain Cas protein,
 Such as Cas9 (type II),
Cas12a (V-A), and Cas13
(type VI)
CRISPR systems can be divided into two classes, six types, and various subtypes.

7. 7

8. 8

9. 9

10. 10

11. 11
1ST thing arise was that
crRNA & tracrRNA are
different entities & jointed
by linker molecule, now
called as single guide RNA
(sgRNA)
This sgRNA can be
synthesised in laboratory
about 20 Nucleotide
Sequence , along with cas
protein , then..
1. Complex forms
2. Complex reads Foreign
DNA
3. Binding occurs
4. DNA cleaved

12. 12

13. 13

14. • Anti-CRISPR (Anti-Clustered Regularly Interspaced Short Palindromic
Repeats or Acr) is a group of proteins found in phages, that inhibit the
normal activity of CRISPR-Cas, the immune system of certain bacteria.
• Microbiologist Joseph Bondy-Denomy discovered Anti-CRISPRs , in 2012.
• All known anti-CRISPRs are small proteins, typically 50–150 amino acids in
size.
• They bear little resemblance to one another in terms of sequence or
structure, suggesting they all evolved independently.
14

15. 15
These mutations, which can be merely a single base pair change,
reduce the binding affinity of the crRNA and allow the invading
phage to escape CRISPR-Cas-mediated destruction.
However, bacteria rapidly acquire new spacers in response
to these escape mutants through a process known as primed
adaptation.
In 2013, the first active counter-mechanism to CRISPR-Cas
immunity, Anti-CRISPR proteins, was identified in phages
that infect Pseudomonas aeruginosa.
Before the identification of ANTI-CRISPR proteins, the only way by
which phages could escape CRISPR-Cas-mediated destruction was
through the acquisition of point mutations .

16. • Acr family proteins are named for the type of CRISPR-Cas
system they block and are numbered in order of discovery.
• As for the naming convention of Acr family proteins, it is
established as follows:
– Firstly, the type of system inhibited,
– Then a numerical value referring to the protein family and
– Finally the source of the specific anti-CRISPR protein.
• For example,
 AcrIIA4 indicates that it’s the fourth Acr protein discovered
that blocks type II-A CRISPR-Cas systems.
16

17. Anti-CRISPR protein families
17

18. • Although CRISPR-Cas offers a cheap, fast, and easy way to make edits in
precise target in genomes and could be game changing for treating genetic
disorders and cancer and for other applications.
• But CRISPR faces safety concerns as cas9, may also end up
mutagenizing other sites you don’t want it to ,therefore scientists have
only limited ways to control the technique.
• As longer a CRISPR system remains active in cells, the more likely it is
to edit parts of a genome other than its intended target, yielding
potentially dangerous side effects.
“Think of any technologies humans use—TVs, computers, microwaves .
. . they all have an On and Off button for a reason,” says Rafael Pinilla
Redondo, a molecular microbiologist at the University of Copenhagen.
“You want to have a way to control these genome-editing technologies,
or else the consequences would potentially be disastrous.”
18

19. • Anti-CRISPRs proteins are highly diverse but most block
CRISPR activity one of three ways:
1. Inhibit DNA binding
Example: AcrIIA4 blocks Cas9’s interaction with the PAM site
and AcrIIC3 causes Cas proteins to dimerize which blocks the
PAM recognition site .
2. Inhibit crRNA loading
Example: AcrIIC2 interactions with Cas9 interferes
and prevents the correct assembly of the crRNA-Cas complex .
3. Inhibiting DNA cleavage
Example: AcrIIC1 binds to the HNH endonuclease domain of
Cas9 and prevents target DNA cleavage.
19

20. 20

21. 21

22. 22

23. Genetically encodable.
Broad spectrum
Easy to use
23
Acr genes can be encoded and delivered on vectors to cells in vivo.
Many Acr proteins, AcrIIA5, AcrIIC1 and AcrVA1, inhibit multiple orthologs of
its target cas complex.
•Acr proteins can easily be integrated into a wide range of in vivo and in vitro systems
•They are also complementary to many existing strategies of regulation.

24. 24

25. 25

26. Antibiotic resistance is a public health problem
that is constantly increasing, because of the
bad use of antibiotics.
Phage therapy consists of the infection of
bacteria using phages, which are much
more specific and cause less side effects
than antibiotics.
Acrs could inhibit the CRISPR-Cas9 system of
some bacteria and allow these phages to
infect bacterial cells without being attacked
by its immune system.
26

27.  A biosensor that couples induction of anti-CRISPR
expression or activity with enhancement of
fluorescence expression was constructed in
Saccharomyces cerevisiae.
 In this case, AcrIIA2 and AcrIIA4 were
transcriptionally induced or post-translationally,
where dCas9::sgRNA complexes are programmed to
constitutively repress green fluorescent protein
(eGFP) transcription, enabling a simple readout to
detect these molecules.
27

28. 28
aca genes repress the acr
transcription in bacteria by
binding to acr promotor, thus
downregulate initially high level
of acr transcription.
Post-transcription regulation
of Acr proteins by miRNA, by
binding to the 3’ site of acr
transcript, thus down regulate
the process.
A ligand inducible Acr protein fused to
Destabilization Domain (DD).
In Presence of shield-1 ,DD is stabilized, & Acr
inhibit Cas9.
In Absence of shield-1 ,DD is unstabilized, & Acr
misfolds & degraded , liberating Cas9.

29. • In 2018, molecular biologist Dominik Niopek and his colleagues
combined an anti-CRISPR with a light-sensitive molecule from
oats as a way to switch the protein on and off using light.
• A photo-controllable AcrIIA4 variant was developed by inserting the
LOV2 (Light, oxygen, or voltage) Domain from Avena sativa
phototrophin-1 into AcrIIA4 surface-exposed loop and optimizing for
functionality.
• In the absence of light, AcrIIA4-LOV2 can bind and inhibit Spycas9.
• While on photoexcitation,the LOV2 domain loses its structural
conformation and causes AcrIIA4 to misfold and lose affinity for
cas9
29

30.  Acr proteins have also been used as lab reagents.
 In one case, CRISPR-Cas9 were detected and quantified using AcrIIA4
as an immobilized capture ligand.
 Fixing AcrIIA4 on glassy carbon electrodes enabled the specific
detection of sgRNA-loaded Cas9 using electrochemical, colorimetric
and fluorescent readouts, which can be used to measure Cas9 delivery
efficacy and persistence in biological samples.
 In another case, Acr proteins were used to facilitate adenoviral vector
production.
 A helper-dependent adenovirus was engineered to express Cas9 for
genome editing and self-cleavage after transduction into cells, thereby
allowing editing but preventing Cas9 persistence.
 To prevent self-cleavage during vector production before transduction,
expression of AcrIIA2 and AcrIIA4 was combined with Cas9 mRNA
down regulation.
30

31. LIMITATIONS
of Acr
proteins
ADDITIONAL
COMPONENT
SLOW
REVERSIBILITY
POTENTIAL
TOXICITY OR
IMMUNOGENICITY
31
 Acr proteins are not cell-
permeant and must be delivered
into cells.
 Genetically encoded acr
sequences may necessitate an
additional vector or increase
vector size.
Inhibition event is not readily
reversible without additional
engineering.
Additional parameters must be
considered in vivo,
 Including Acr protein stability,
optimal expression levels, and
potential for off-target
interactions.

32. • One limitation of most anti-CRISPRs is that each
molecule can suppress just one Cas protein at a time.
• But in 2019, a Chinese team and a US group each
discovered anti-CRISPR enzymes that could disable
multiple copies of a Cas protein by slicing up their
guide RNAs, by using them to quickly shut down all
Cas9 proteins.
32

33. • Acr of type VI of CRISPR-cas system yet to be discovered.
• Present research on CRISPR-Acr interactions demonstrated that the
initial density of Acr-phages that infect bacteria with CRISPR resistance
determines whether phages go extinct or amplify??
• It has been found to have long-term suppression of CRISPR resistance
following an unsuccessful infection, which is consistent with the slow
dissociation kinetics of Acr-Cas protein complexes.
• If this critical threshold is not reached, the Acr-phages go extinct, and
immuno suppressed hosts revert to their resistant state .
33

34.  Although CRISPR-Cas offers a cheap, fast, and easy way to make edits in precise
target in genomes , as CRISPR faces safety concerns because there are limited
ways to control the technique.
 Anti-CRISPR (Anti-Clustered Regularly Interspaced Short Palindromic Repeats or
Acr) is a group of proteins found in phages, that inhibit the normal activity
of CRISPR-Cas, the immune system of certain bacteria.
 Before the discovery of this type of family proteins, the acquisition of mutations
was the only way known that phages could use to avoid CRISPR-Cas mediated
shattering, by reducing the binding affinity of the phage and CRISPR.
 Nonetheless, bacteria have mechanisms to retarget the mutant bacteriophage, a
process that it is called "priming adaptation".
 So, as far as researchers currently know, anti-CRISPR is the most effective way to
ensure the survival of phages throughout the infection process of bacteria.
 Although it has many limitation like slow reversibility , toxicty etc. ,which need
more future research to be made.
34

35.  Hoffmann MD, Mathony J, Upmeier zu Belzen J, Harteveld Z,
Aschenbrenner S, Stengl C, Grimm D, Correia BE, Eils R, Niopek D.
(2021) Optogenetic control of Neisseria meningitidis Cas9 genome editing
using an engineered, light-switchable anti-CRISPR protein. Nucleic acids
res 18: 49
 Marino ND, Pinilla-Redondo R, Csorgo B, Bondy-Denomy J (2020) Anti-
CRISPR protein applications: natural brakes for CRISPR-Cas technologies.
Nat methods 17(5): 471-9.
 Liu Q, Zhang H, Huang X (2020) Anti‐CRISPR proteins targeting the
CRISPR‐Cas system enrich the toolkit for genetic engineering. The FEBS J
287(4) :626-44.
 Lee J. Anti-CRISPR Proteins: Applications in Genome Engineering.
 VEDIO - https://youtu.be/2pp17E4E-O8
35

36. 36