Direct observation of DNA target searching and cleavage by CRISPR-Cas12a

Yongmoon Jeon, You Hee Choi, Yunsu Jang, Jihyeon Yu, Jiyoung Goo, Gyejun Lee, You Kyeong Jeong, Seung Hwan Lee, In San Kim, Jin Soo Kim, Cherlhyun Jeong, Sanghwa Lee, Sangsu Bae

Research output: Contribution to journalArticlepeer-review

118 Citations (Scopus)

Abstract

Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.

Original languageEnglish
Article number2777
JournalNature communications
Volume9
Issue number1
DOIs
Publication statusPublished - 2018 Dec 1

Bibliographical note

Funding Information:
We thank Prof. J.-B. Lee (POSTECH) for the helpful advice and discussion. This work was supported by National Research Foundation of Korea (NRF) Grants (No. 2017R1A6A3A01007932 to Yo.J.; 2016R1A6A3A11930747 to Y.H.C.; 2017R1A3B1023418 and 2018R1A2B2008995 to C.J.; 2015R1C1A1A01055164 and 2016R1A5A1007318 to S.L.; 2017M3A9G8084539 and 2018M3A9H3022412 to S.B.), and by a grant from the KIST Institutional Program to C.J., and by a grant from the National R&D Program for Cancer Control of Ministry (1520090), the GIST Research Insitute to S.L., and by grants from Next Generation BioGreen 21 Program (PJ01319301), Korea Healthcare technology R&D Project (HI16C1012) to S.B.

Publisher Copyright:
© 2018 The Author(s).

ASJC Scopus subject areas

  • General
  • General Physics and Astronomy
  • General Chemistry
  • General Biochemistry,Genetics and Molecular Biology

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