Conformational Dynamics of CasX (Cas12e) in Mediating DNA Cleavage Revealed by Single-molecule FRET

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2024 Jul 12

PMID 38994558

Authors

Wenjing Xing

Danyuan Li

Wenjuan Wang

Jun-Jie Gogo Liu

Chunlai Chen

Affiliations

Soon will be listed here.

Abstract

CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared to the widely used Cas9 and Cas12a. Although the structures of CasX-sgRNA-DNA ternary complexes have been resolved and uncover a distinctive NTSB domain, the dynamic behaviors of CasX are not well characterized. In this study, we employed single-molecule and biochemical assays to investigate the conformational dynamics of two CasX homologs, DpbCasX and PlmCasX, from DNA binding to target cleavage and fragment release. Our results indicate that CasX cleaves the non-target strand and the target strand sequentially with relative irreversible dynamics. The two CasX homologs exhibited different cleavage patterns and specificities. The dynamic characterization of CasX also reveals a PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate the mechanistic basis for why modification of sgRNA and the NTSB domain can affect its activity. Interestingly, CasX has less effective target search efficiency than Cas9 and Cas12a, potentially accounting for its lower genome editing efficiency. This observation opens a new avenue for future protein engineering.

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References

Xin C, Yin J, Yuan S, Ou L, Liu M, Zhang W . Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption. Nat Commun. 2022; 13(1):5623. PMC: 9509373. DOI: 10.1038/s41467-022-33346-1. View

Singh D, Mallon J, Poddar A, Wang Y, Tippana R, Yang O . Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a). Proc Natl Acad Sci U S A. 2018; 115(21):5444-5449. PMC: 6003496. DOI: 10.1073/pnas.1718686115. View

Jones D, Leroy P, Unoson C, Fange D, Curic V, Lawson M . Kinetics of dCas9 target search in . Science. 2017; 357(6358):1420-1424. PMC: 6150439. DOI: 10.1126/science.aah7084. View

Makarova K, Wolf Y, Iranzo J, Shmakov S, Alkhnbashi O, Brouns S . Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2019; 18(2):67-83. PMC: 8905525. DOI: 10.1038/s41579-019-0299-x. View

Tu M, Lin L, Cheng Y, He X, Sun H, Xie H . A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells. Nucleic Acids Res. 2017; 45(19):11295-11304. PMC: 5737432. DOI: 10.1093/nar/gkx783. View

Toth E, Czene B, Kulcsar P, Krausz S, Talas A, Nyeste A . Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants. Nucleic Acids Res. 2018; 46(19):10272-10285. PMC: 6212782. DOI: 10.1093/nar/gky815. View

Gorski S, Vogel J, Doudna J . RNA-based recognition and targeting: sowing the seeds of specificity. Nat Rev Mol Cell Biol. 2017; 18(4):215-228. DOI: 10.1038/nrm.2016.174. View

Globyte V, Lee S, Bae T, Kim J, Joo C . CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion. EMBO J. 2018; 38(4). PMC: 6376262. DOI: 10.15252/embj.201899466. View

Rostain W, Grebert T, Vyhovskyi D, Thiel Pizarro P, Tshinsele-Van Bellingen G, Cui L . Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity. Nucleic Acids Res. 2023; 51(7):3485-3496. PMC: 10123097. DOI: 10.1093/nar/gkad170. View

10.

Wright A, Nunez J, Doudna J . Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering. Cell. 2016; 164(1-2):29-44. DOI: 10.1016/j.cell.2015.12.035. View

11.

Kleinstiver B, Pattanayak V, Prew M, Tsai S, Nguyen N, Zheng Z . High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016; 529(7587):490-5. PMC: 4851738. DOI: 10.1038/nature16526. View

12.

Kim D, Luk K, Wolfe S, Kim J . Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases. Annu Rev Biochem. 2019; 88:191-220. DOI: 10.1146/annurev-biochem-013118-111730. View

13.

Karpov D, Demidova N, Kulagin K, Shuvalova A, Kovalev M, Simonov R . Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems. Int J Mol Sci. 2022; 23(23). PMC: 9738314. DOI: 10.3390/ijms232314847. View

14.

Ran F, Cong L, Yan W, Scott D, Gootenberg J, Kriz A . In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-91. PMC: 4393360. DOI: 10.1038/nature14299. View

15.

Zeng Y, Cui Y, Zhang Y, Zhang Y, Liang M, Chen H . The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex. Nucleic Acids Res. 2017; 46(1):350-361. PMC: 5758904. DOI: 10.1093/nar/gkx1117. View

16.

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

17.

Zetsche B, Gootenberg J, Abudayyeh O, Slaymaker I, Makarova K, Essletzbichler P . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163(3):759-71. PMC: 4638220. DOI: 10.1016/j.cell.2015.09.038. View

18.

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

19.

Kim N, Kim H, Lee S, Seo J, Choi J, Park J . Prediction of the sequence-specific cleavage activity of Cas9 variants. Nat Biotechnol. 2020; 38(11):1328-1336. DOI: 10.1038/s41587-020-0537-9. View

20.

Knott G, Doudna J . CRISPR-Cas guides the future of genetic engineering. Science. 2018; 361(6405):866-869. PMC: 6455913. DOI: 10.1126/science.aat5011. View