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Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins

Abstract

CRISPR loci and Cas proteins provide adaptive immunity in prokaryotes against invading bacteriophages and plasmids. In response, bacteriophages have evolved a broad spectrum of anti-CRISPR proteins (anti-CRISPRs) to counteract and overcome this immunity pathway. Numerous anti-CRISPRs have been identified to date, which suppress single-subunit Cas effectors (in CRISPR class 2, type II, V and VI systems) and multisubunit Cascade effectors (in CRISPR class 1, type I and III systems). Crystallography and cryo-electron microscopy structural studies of anti-CRISPRs bound to effector complexes, complemented by functional experiments in vitro and in vivo, have identified four major CRISPR–Cas suppression mechanisms: inhibition of CRISPR–Cas complex assembly, blocking of target binding, prevention of target cleavage, and degradation of cyclic oligonucleotide signalling molecules. In this Review, we discuss novel mechanistic insights into anti-CRISPR function that have emerged from X-ray crystallography and cryo-electron microscopy studies, and how these structures in combination with function studies provide valuable tools for the ever-growing CRISPR–Cas biotechnology toolbox, to be used for precise and robust genome editing and other applications.

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Fig. 1: Functional mechanisms of type I anti-CRISPRs.
Fig. 2: Functional mechanisms of type III anti-CRISPRs.
Fig. 3: Functional mechanisms of type II anti-CRISPRs.
Fig. 4: Functional mechanisms of type V anti-CRISPRs.
Fig. 5: Functional mechanisms of type VI anti-CRISPRs.
Fig. 6: Anti-CRISPR biotechnology applications.

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Acknowledgements

The research was supported by NIH grant GM129430 to D.J.P. and by the Memorial Sloan Kettering Cancer Center Core Grant P30 CA008748.

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N.J. wrote the first draft of the Review. Both authors contributed to several rounds of manuscript editing.

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Correspondence to Ning Jia or Dinshaw J. Patel.

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D.J.P. is a consultant for Ventus Therapeutics. N.J. declares no competing interests.

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Nature Reviews Molecular Cell Biology thanks Joseph Bondy-Denomy, Zhiwei Huang and Min Wu for their contribution to the peer review of this work.

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Supplementary information

Glossary

Restriction–modification

A system in prokaryotes that typically contains a restriction endonuclease and a methylase that target pathogenic DNA.

Second messenger

An intracellular signalling molecule that transmits and amplifies the signal generated by extracellular signalling molecules (the first messengers).

R-loop

An RNA–DNA hybrid structure in which RNA pairs with a complementary DNA strand, causing the non-complementary DNA strand to loop out.

Primed spacer acquisition

The preferential acquisition of new spacers from a target recognized by a CRISPR-derived RNA-guided effector complex.

CRISPR interference

(CRISPRi). A technique that typically exploits a catalytically dead CRISPR–Cas effector complex to block transcription and thereby silence genes.

Single guide RNA

(sgRNA). A synthetic fusion of the trans-activating CRISPR RNA (tracrRNA) and the CRISPR-derived RNA (crRNA), which functions similarly to the native crRNA–tracrRNA duplex in guiding Cas9 to a DNA target site.

Gene drive

A genetic engineering technique for rapidly spreading a particular suite of genes through a population.

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Jia, N., Patel, D.J. Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins. Nat Rev Mol Cell Biol 22, 563–579 (2021). https://doi.org/10.1038/s41580-021-00371-9

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