Temperature-Responsive Competitive Inhibition of CRISPR-Cas9

Overview

Journal Mol Cell

Publisher Cell Press

Specialty Cell Biology

Date 2019 Jan 1

PMID 30595438

Citations 38

Authors

Fuguo Jiang

Jun-Jie Liu

Beatriz A Osuna

Michael Xu

Joel D Berry

Benjamin J Rauch

Eva Nogales

Joseph Bondy-Denomy

Jennifer A Doudna

Affiliations

Soon will be listed here.

Abstract

CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six inhibitors (AcrIIA1-AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally.

Citing Articles

Current Updates of CRISPR/Cas System and Anti-CRISPR Proteins: Innovative Applications to Improve the Genome Editing Strategies.

Allemailem K, Almatroudi A, Alrumaihi F, Alradhi A, Theyab A, Algahtani M Int J Nanomedicine. 2024; 19:10185-10212.

PMID: 39399829 PMC: 11471075. DOI: 10.2147/IJN.S479068.

Inhibition mechanisms of CRISPR-Cas9 by AcrIIA25.1 and AcrIIA32.

Zheng J, Zhu Y, Huang T, Gao W, He J, Huang Z Sci China Life Sci. 2024; 67(9):1781-1791.

PMID: 38842649 DOI: 10.1007/s11427-024-2607-8.

An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding.

Deng X, Sun W, Li X, Wang J, Cheng Z, Sheng G Nat Commun. 2024; 15(1):1806.

PMID: 38418450 PMC: 10901769. DOI: 10.1038/s41467-024-45987-5.

Conformational plasticity of SpyCas9 induced by AcrIIA4 and AcrIIA2: Insights from molecular dynamics simulation.

Wen S, Zhao Y, Qi X, Cai M, Huang K, Liu H Comput Struct Biotechnol J. 2024; 23:537-548.

PMID: 38235361 PMC: 10791570. DOI: 10.1016/j.csbj.2023.12.030.

Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs.

Kawamata M, Suzuki H, Kimura R, Suzuki A Nat Biomed Eng. 2023; 7(5):672-691.

PMID: 37037965 PMC: 10195680. DOI: 10.1038/s41551-023-01011-7.

References

Rauch B, Silvis M, Hultquist J, Waters C, McGregor M, Krogan N . Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. Cell. 2017; 168(1-2):150-158.e10. PMC: 5235966. DOI: 10.1016/j.cell.2016.12.009. View

Tang G, Peng L, Baldwin P, Mann D, Jiang W, Rees I . EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol. 2006; 157(1):38-46. DOI: 10.1016/j.jsb.2006.05.009. View

Koonin E, Makarova K, Zhang F . Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol. 2017; 37:67-78. PMC: 5776717. DOI: 10.1016/j.mib.2017.05.008. View

Marraffini L, Sontheimer E . CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet. 2010; 11(3):181-90. PMC: 2928866. DOI: 10.1038/nrg2749. View

Hynes A, Rousseau G, Agudelo D, Goulet A, Amigues B, Loehr J . Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. Nat Commun. 2018; 9(1):2919. PMC: 6060171. DOI: 10.1038/s41467-018-05092-w. View

Wiedenheft B, van Duijn E, Bultema J, Bultema J, Waghmare S, Waghmare S . RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci U S A. 2011; 108(25):10092-7. PMC: 3121849. DOI: 10.1073/pnas.1102716108. View

Pawluk A, Davidson A, Maxwell K . Anti-CRISPR: discovery, mechanism and function. Nat Rev Microbiol. 2017; 16(1):12-17. DOI: 10.1038/nrmicro.2017.120. View

Jiang F, Zhou K, Ma L, Gressel S, Doudna J . STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition. Science. 2015; 348(6242):1477-81. DOI: 10.1126/science.aab1452. View

Makarova K, Wolf Y, Alkhnbashi O, Costa F, Shah S, Saunders S . An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015; 13(11):722-36. PMC: 5426118. DOI: 10.1038/nrmicro3569. View

10.

Grant G . The ACT domain: a small molecule binding domain and its role as a common regulatory element. J Biol Chem. 2006; 281(45):33825-9. DOI: 10.1074/jbc.R600024200. View

11.

Hale C, Zhao P, Olson S, Duff M, Graveley B, Wells L . RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell. 2009; 139(5):945-56. PMC: 2951265. DOI: 10.1016/j.cell.2009.07.040. View

12.

Basgall E, Goetting S, Goeckel M, Giersch R, Roggenkamp E, Schrock M . Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae. Microbiology (Reading). 2018; 164(4):464-474. PMC: 5982135. DOI: 10.1099/mic.0.000635. View

13.

Hille F, Charpentier E . CRISPR-Cas: biology, mechanisms and relevance. Philos Trans R Soc Lond B Biol Sci. 2016; 371(1707). PMC: 5052741. DOI: 10.1098/rstb.2015.0496. View

14.

Kimanius D, Forsberg B, Scheres S, Lindahl E . Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. Elife. 2016; 5. PMC: 5310839. DOI: 10.7554/eLife.18722. View

15.

Marraffini L . CRISPR-Cas immunity in prokaryotes. Nature. 2015; 526(7571):55-61. DOI: 10.1038/nature15386. View

16.

Dong D, Guo M, Wang S, Zhu Y, Wang S, Xiong Z . Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein. Nature. 2017; 546(7658):436-439. DOI: 10.1038/nature22377. View

17.

Anders C, Niewoehner O, Duerst A, Jinek M . Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014; 513(7519):569-73. PMC: 4176945. DOI: 10.1038/nature13579. View

18.

Rohou A, Grigorieff N . CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J Struct Biol. 2015; 192(2):216-21. PMC: 6760662. DOI: 10.1016/j.jsb.2015.08.008. View

19.

Punjani A, Rubinstein J, Fleet D, Brubaker M . cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods. 2017; 14(3):290-296. DOI: 10.1038/nmeth.4169. View

20.

Chen V, Arendall 3rd W, Headd J, Keedy D, Immormino R, Kapral G . MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 1):12-21. PMC: 2803126. DOI: 10.1107/S0907444909042073. View