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Structural biology of CRISPR–Cas immunity and genome editing enzymes

Abstract

CRISPR–Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR–Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR–Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR–Cas systems and mobile genetic elements, including the origins and evolution of CRISPR–Cas systems from DNA transposons, retrotransposons and toxin–antitoxin modules. We discuss how the evolution and structural diversity of CRISPR–Cas systems explain their functional complexity and utility as genome editing tools.

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Fig. 1: CRISPR–Cas systems provide bacteria and archaea with adaptive immunity.
Fig. 2: Modular organization of CRISPR–Cas systems.
Fig. 3: Evolution and diversity of the CRISPR integrase architecture.
Fig. 4: Mechanisms of CRISPR prespacer biogenesis and orientation.
Fig. 5: Mechanism of Cas9.
Fig. 6: Mechanism of Cas12.
Fig. 7: Cas-mediated transposition.

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Acknowledgements

The authors thank J. C. Cofsky, K. M. Soczek and G. J. Knott for sharing the coordinates of Cas9 bound to linear and bent double-stranded DNA before publication. J.Y.W. is supported by the US National Science Foundation Graduate Fellowship and was previously supported by the Berkeley Graduate Fellowship. P.P. receives funding from the European Regional Development Fund under grant agreement number 01.2.2-CPVA-V-716-01-0001 with the Central Project Management Agency (CPVA), Lithuania, and from the Research Council of Lithuania (LMTLT) under grant agreement number S-MIP-22-10. This material is based upon work supported by the US National Science Foundation under award number 1817593 and by the Somatic Cell Genome Editing Program of the Common Fund of the US National Institutes of Health under award number U01AI142817-02. J.A.D. is a Howard Hughes Medical Institute investigator. The authors thank G. J. Knott and A. Lapinaite for helpful discussions.

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Correspondence to Patrick Pausch or Jennifer A. Doudna.

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J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Vertex, Caribou Biosciences, Intellia Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Synthego, Algen Biotechnologies, Felix Biosciences, The Column Group and Inari. J.A.D. is the Chief Science Advisor of Sixth Street, is on the Board of Directors at Altos, Johnson & Johnson and Tempus, and has research projects sponsored by Biogen, Pfizer, AppleTree Partners and Roche. The Regents of the University of California have patents issued and pending for CRISPR technologies on which P.P. and J.A.D. are named as inventors. J.Y.W. declares no competing interests.

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Wang, J.Y., Pausch, P. & Doudna, J.A. Structural biology of CRISPR–Cas immunity and genome editing enzymes. Nat Rev Microbiol 20, 641–656 (2022). https://doi.org/10.1038/s41579-022-00739-4

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