The last decade has seen the fields of molecular biology and genetics transformed by the development of CRISPR-based gene editing technologies. These technologies were derived from bacterial defense systems that protect against viral invasion. In the past few years, a variety of phages and other mobile genetic elements have been shown to encode anti-CRISPR proteins (Acrs) that interact directly (in a sequence-independent manner) with components of the CRISPR-Cas system and inactivate it. The discovery of anti-CRISPR proteins has opened up a new area of phage research and has provided a valuable addition to the CRISPR toolbox as an ‘off switch’’ for Cas9 activity. But, most of the CRISPR-Cas systems still have no known inhibitors, suggesting that many anti-CRISPR protein families are yet to be discovered which can be used as regulators for genome engineering and other biotechnological applications.

1. Anti-CRISPR mediated control of
gene editing
Roshni. M,
PALB6078
PhD Plant Biotechnology

2. “He that wrestles with us strengthen our nerves and sharpens our skill.
Our antagonist is our helper”
Edmund Burke
3/11/2021 Dept. of Plant Biotechnology 2

3. Outline of the presentation
 Introduction
 Phage evasion
 Identification and diversity of anti-CRISPR
 Methods of anti-CRISPR discovery
 Biological relevance of Acrs
 Types of Acrs
 Mechanism
 Case studies
 Application of anti-CRISPR
 Future prospects
 Conclusion
3/11/2021 Dept. of Plant Biotechnology 3

4. Toxin –
antitoxin
BREX
DISARM
Restriction
modification
Super
infection
exclusion
Abortive
infection
Receptor
masking
Innate
Bacterial defense Strategies
Counter
defense
by
Phage
Introduction
(Trasanidou et al., 2019)
Red queen hypothesis
Is it unusual to defend and counter- defend ?
Answer is “ No”
(Valen, 1973; Forterre and Prangishvili, 2009)
• Natural phenomenon
• Perennial arms race - Co-evolution
• To survive in the changing environment
3/11/2021 Dept. of Plant Biotechnology 4

5. Denomy et al., 2013
ANTI- CRISPR
???
CRISPR
Adaptive and heritable immunity
Yes !!!
Bacteriophages impose a significant selective
pressure -1023 phage infections every second
3/11/2021 Dept. of Plant Biotechnology 5

6. • CRISPR immune
system
Bacteria
• Mutational drift
CRISPR escape
strategy • Point mutations
in PAM or seed
region
Phage
Fitness cost ???
Long term survival ?
Bacteria Phage
Acquire more spacers Extinction
Phage evasion
Denomy et al., 2015
3/11/2021 Dept. of Plant Biotechnology 6

7. ANTI-CRIPSR VIDEO
CRISPR proteins and their widespread applications
3/11/2021 Dept. of Plant Biotechnology 7

8. Acrs
Type I-F and type I-E Acrs
Pseudomonas aeruginosa
AcrF6 Homologue
Gammaproteobacteria
Type IIC
Brackiella odipodis Neisseria meningitidis
AcrDI
Sulfolobus islandicus (archael virus)
Identification of anti CRISPR proteins and diversity
He et al., 2018
Anti-CRISPRs are abbreviated as Acrs
3/11/2021 Dept. of Plant Biotechnology 8

9. Anti-CRISPR genes across
bacterial phylogeny
Diversity and types of CRISPR-Cas
interference proteins
(Pawluk et al., 2017)
3/11/2021 Dept. of Plant Biotechnology 9

10. A comprehensive online resource for anti-CRISPR proteins
• 432 anti-CRISPR proteins
• protein sequences, DNA sequences, coding regions, source
organisms, taxonomy, virulence, and protein interactors and the
corresponding three-dimensional structures
Anti CRISPR db
(http://cefg.uestc.cn/anti-CRISPRdb)
3/11/2021 Dept. of Plant Biotechnology 10

11. Methods of anti-CRISPR discovery
1. Functional assay with Pseudomonas aeruginosa lysogens
Prophages supporting the replication of CRISPR- Cas targeted phages
2. Guilt-by-association bioinformatics approach
Experimentally validated anti-CRISPR genes found in association with aca1 gene
PSI blast- 95 % sequence identity in aca gene
3. Self-targeting bioinformatic approach
CRISPR- Coexistence of a prophage and targeting spacer
4. Functional screen with immunized bacteria
(Stanley and Maxwelll, 2018)
Bacterial strain is immunized with spacers
3/11/2021 Dept. of Plant Biotechnology 11

12. Biological relevance of anti-CRISPR in phage
• Anti immune protein inhibitors
• Accessory genes in phage- not essential for lytic or
lysogenic replication of phage
• Conditionally essential
• Increase the fitness of phage population- reflects its
adaptation to a specific host
(Borges et al., 2017)
3/11/2021 Dept. of Plant Biotechnology 12

13. • Bacteria with CRISPR immunity-
partially resistant to Acr-phage
• Sequentially infecting Acr-phages
cooperate to overcome CRISPR
resistance
• Acr-phage epidemiology depends
on the initial phage density
• CRISPR-resistant bacteria can
drive Acr-phages extinct
Landsberger et al., 2018
3/11/2021 Dept. of Plant Biotechnology 13

14. • Anti-CRISPR genes are transcribed
to high levels quickly after phage
infection
• Aca proteins are ubiquitous DNA-
binding proteins encoded in anti-
CRISPR operons
• Aca proteins repress anti-CRISPR
transcription
• Aca function obviates deleterious
effects of high anti- CRISPR
transcription
Stanley et al., 2019
3/11/2021 Dept. of Plant Biotechnology 14

15. Trasanidou et al., 2019
Mechanism of Acrs
3/11/2021 Dept. of Plant Biotechnology 15

16. Class 1 Acr
a. Preventing DNA
binding via interaction
with cascade complex
b. Preventing DNA
cleavage via interaction
with Cas3 nuclease
c. Preventing DNA
binding via interaction
with cas9 protein
catalytic site
d. Preventing CRISPR-
Cas immunity via
putative binding to RNA
or DNA molecules
a1. Steric occlusion of
DNA binding
a2. DNA mimic
b1. Disruption of binding to
the cascade:dsDNA chimera
a1. Eg: AcrF1
Results in conformational change that
sterically blocks access of crguide RNA
to the target DNA
a2 Eg: AcrF2
Small acidic protein behaves as a DNA
mimic and interact with Cas5f: Cas8f
heterodimer and shoves off the hook
b1 Eg: AcrF3
Forms homodimer binding to Cas3 nuclease
and blocks the interaction sites
Locks ATP dependent into inactive form of Cas3
nuclease
b1 Eg: AcrIIA2, A4, C2 , C1
Associate with PAM interacting, Topo-
homology, RuvC, WED, HNH and Rec2 domain
Dimerization of Cas9, DNA mimic.
d1 Eg: AcrIIA1 recognize and associate
with heterogenous RNA
Class 2 Acr
Stanley and Maxwelll, 2018
3/11/2021 Dept. of Plant Biotechnology 16

17. Why specifically inhibit interference step?
• Bacterial cell will be ready to protect itself from phage
• Bacteria upregulate CRISPR Cas expression when encountered
with high cell density of phage
• Acrs can inhibit both preassembled complex and
ribonucleoprotein surveillance complex
3/11/2021 Dept. of Plant Biotechnology 17

18. Case studies
3/11/2021 Dept. of Plant Biotechnology 18

19. Objective
Characterization of Acrs as a tool for temporal and inhibitory
control of CRISPR genetic circuits in range of contexts (cell lines,
yeast and mammalian cells)
3/11/2021 Dept. of Plant Biotechnology 19

20. Materials and Results
1. Acrs inhibit CRISPR-based gene regulation in mammalian cells
• Acrs inhibiting CRISPRa and CRISPRi on reporter genes
• Reporter cell line – HEK239T with GFP
• AcrIIA4 – significant reduction in CRISPR gene regulation
Representative raw fluorescence flow cytometry data with summary comparison
3/11/2021 Dept. of Plant Biotechnology 20

21. 2. Acrs can inhibit CRISPRa and CRISPRi on endogenous genes
• Doxycycline induced PiggyBac constructs
• sgRNA targetted to endogenous CXCR4 chemokine receptor
• Tested in both HEK239T and hiPSC (human induced Pluripotent
Stem Cells )
b. Change in the mean expression of CXCR4 in the presence and
absence of Acrs
d. Endogenous gene regulation in hiPSC
3/11/2021 Dept. of Plant Biotechnology 21

22. 3. Acrs inhibit CRISPR activity in yeast cells
Tested for the activity of Acrs in both Cas9 and dCas9 system
Multiple type II Acrs found effective in Cas9 experiment
Acr induced change in reporter expression in CRISPRa and CRISPRi experiments
3/11/2021 Dept. of Plant Biotechnology 22

23. 4. Acrs negate activity of CRISPR-based synthetic devices
a. Inclusion of Acrs in Cha-Cha system c. Shield 1 inducible experiment for dosage
response
b. Change in fluorescence in with various linkers d. dosage response
3/11/2021 Dept. of Plant Biotechnology 23

24. 5. Acrs offer a means to genomically write-protected cells- human genome
editing
T7E1 and TIDE assay comparing editing efficiency
3/11/2021 Dept. of Plant Biotechnology 24

25. 6. Acr-based genetic circuits provide pulsatile gene expression
a. Preprogrammed Acr based circuits
b. Cell tracking of IFFL circuits
c. GFP production in IFFL circuit – density plot
(pulsative)
3/11/2021 Dept. of Plant Biotechnology 25

26. CRISPRa
Acr
IFFL circuit
GFP trajectory
3/11/2021 Dept. of Plant Biotechnology 26

27. • Proof-of principle demonstration -integrating Acrs in the
genome to generate cells that are immune to unlicensed
editing applications.
• Effective internal control switches for CRISPR genetic circuit
• Evaluated and characterized anti-CRISPR proteins (Cas9 and
dCas9 system) using both CRISPRi and CRISPRa in mammalian
cells.
• AcrIIA4 is a potent regulator of (d)Cas9 activity in a wide
variety of contexts (reporter or endogenous genes) and cell
types (HEK293T, hiPSC, and yeast).
Key findings of the paper
3/11/2021 Dept. of Plant Biotechnology 27

28. Objective
• Using bioinformatics approach to identify the putative anti
CRISPR proteins outside aeruginosa cluster – “guilt by
association studies”
• To test the dual anti-CRISPR activity of AcrF6 and identify the
homologues
Case study 2
3/11/2021 Dept. of Plant Biotechnology 28

29. Discovery and characterization of aca1-associated anti-CRISPR genes
P. aeruginosa strains used - UCBPP-PA14 for type I-F and SMC4386 for type I-E
P. aeruginosa
bacteriophage
Plaque spotting
experiment
PSI blast
Materials and Results
3/11/2021 Dept. of Plant Biotechnology 29

30. Investigation of AcrF6Pae for dual anti-CRISPR activity
Mutant studies
35 %
27%
Biological activity
3/11/2021 Dept. of Plant Biotechnology 30

31. Discovery and characterization of aca2-associated anti-CRISPR genes
Genomic contexts
3/11/2021 Dept. of Plant Biotechnology 31

32. Newly identified anti-CRISPRs impact diverse type I-F CRISPR-Cas systems
Gene arrangement and pairwise percent identities for
type I-F Cas proteins
Relative plaquing efficiency
Sequence similarity dendrogram
3/11/2021 Dept. of Plant Biotechnology 32

33. Key findings of the paper
• First evidence that Acrs were widespread in
Gammaproteobacteria other than P. aeruginosa
• Identified 5 new families of Acrs by this approach
• Acrs are found in varied genomic contexts
• Acrs have broad specificity but needs to be experimentally
validated
3/11/2021 Dept. of Plant Biotechnology 33

34. Applications of Anti-CRISPR
• Phage host interactions
• Prophylactic measure for biosecurity and unintended
use of CRISPR editing
• Temporal, spatial and conditional control of CRISPR- Cas
as “off switches”
• Can be preprogrammed in the synthetic CRISPR circuits
• Effectively control the spread of gene drive
• Phage therapy
3/11/2021 Dept. of Plant Biotechnology 34

35. Evaluation of anti-CRISPRS in Plant genome editing ?
Future prospects
Using anti-CRISPR proteins as internal nodes in the plant CRISPR circuits
Anti-CRISPR analysis can be done by transient transfection studies
3/11/2021 Dept. of Plant Biotechnology 35

36. • Percent inhibition by anti CRISPR, how it influence the
phage and bacterial evolution and fitness
• Where were these diverse genes acquired from, and how
did they evolve?
• How efficient it can be used as off-switch for CRISPR Cas
Conclusion
3/11/2021 Dept. of Plant Biotechnology 36

37. Thank you
3/11/2021 Dept. of Plant Biotechnology 37