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 () and Sangsu Bae ()
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Yongmoon Jeon: Korea Institute of Science and Technology
You Hee Choi: Gwangju Institute of Science and Technology
Yunsu Jang: Gwangju Institute of Science and Technology
Jihyeon Yu: Hanyang University
Jiyoung Goo: Korea Institute of Science and Technology
Gyejun Lee: Korea Institute of Science and Technology
You Kyeong Jeong: Hanyang University
Seung Hwan Lee: Institute for Basic Science
In-San Kim: Korea Institute of Science and Technology
Jin-Soo Kim: Institute for Basic Science
Cherlhyun Jeong: Korea Institute of Science and Technology
Sanghwa Lee: Gwangju Institute of Science and Technology
Sangsu Bae: Hanyang University

Nature Communications, 2018, vol. 9, issue 1, 1-11

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-05245-x

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