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

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27 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

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)

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    Jeon, Y., Choi, Y. H., Jang, Y., Yu, J., Goo, J., Lee, G., Jeong, Y. K., Lee, S. H., Kim, I. S., Kim, J. S., Jeong, C., Lee, S., & Bae, S. (2018). Direct observation of DNA target searching and cleavage by CRISPR-Cas12a. Nature communications, 9(1), [2777]. https://doi.org/10.1038/s41467-018-05245-x