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.
2. “He that wrestles with us strengthen our nerves and sharpens our skill.
Our antagonist is our helper”
Edmund Burke
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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
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5. Denomy et al., 2013
ANTI- CRISPR
???
CRISPR
Adaptive and heritable immunity
Yes !!!
Bacteriophages impose a significant selective
pressure -1023 phage infections every second
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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
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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
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9. Anti-CRISPR genes across
bacterial phylogeny
Diversity and types of CRISPR-Cas
interference proteins
(Pawluk et al., 2017)
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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)
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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
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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)
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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
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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
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15. Trasanidou et al., 2019
Mechanism of Acrs
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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
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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
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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)
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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
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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
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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
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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
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24. 5. Acrs offer a means to genomically write-protected cells- human genome
editing
T7E1 and TIDE assay comparing editing efficiency
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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)
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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
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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
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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
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30. Investigation of AcrF6Pae for dual anti-CRISPR activity
Mutant studies
35 %
27%
Biological activity
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31. Discovery and characterization of aca2-associated anti-CRISPR genes
Genomic contexts
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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
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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
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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
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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
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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
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Schematic overview of the different Acrs and their mechanisms. The green boxes on the left show the different stages of CRISPR-Cas immunity. The columns
indicate which CRISPR-Cas type is suppressed by which (group of) Acrs. Acrs are depicted as circles with their abbreviated names (e.g. AcrIF3 is abbreviated to IF3).
A dashed line indicates a suggested role for the particular Acr or that the Acr mechanism remains to be elucidated. Note that most Acrs appear to suppress the
interference stage, whereas only one Acr (AcrIF3) suppressed different stages.