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

Yongmoon Jeon1, You Hee Choi2,3, Yunsu Jang2,3

  • 1Center for Theragnosis, Korea Institute of Science and Technology, Seoul, 02792, South Korea.

Nature Communications
|July 19, 2018
PubMed

Insights

This study reveals how CRISPR-Cas12a (also known as Cpf1) finds and cuts DNA. It moves along DNA and cleaves strands sequentially, with the PAM sequence playing a minor role in unwinding.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • CRISPR-Cas12a (Cpf1) is a versatile genome editing tool distinct from Cas9.
  • The precise mechanisms of Cas12a DNA target searching and cleavage remain incompletely understood.

Purpose of the Study:

  • To elucidate the detailed molecular mechanisms of DNA target recognition and cleavage by Cas12a.
  • To compare the functional role of the protospacer-adjacent motif (PAM) in Cas12a activity versus Cas9.

Main Methods:

  • Single-molecule fluorescence assays were employed to directly observe the behavior of Acidaminococcus sp. Cas12a (AsCas12a).
  • Real-time observation of ribonucleoprotein complex interactions with DNA targets.

Main Results:

  • AsCas12a ribonucleoproteins exhibit one-dimensional diffusion along DNA to locate target sites.
  • DNA cleavage occurs in a specific order: the non-target strand is cleaved first, followed by the target strand.
  • The AsCas12a PAM contributes minimally to DNA unwinding during R-loop formation and has a negligible role in DNA cleavage.

Conclusions:

  • Cas12a utilizes a distinct DNA search strategy involving 1D diffusion.
  • The sequential cleavage mechanism and limited PAM role differentiate Cas12a from Cas9.
  • These findings provide crucial insights into the mechanistic basis of Cas12a-mediated genome editing.

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