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

Author

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  • Yongmoon Jeon

    (Korea Institute of Science and Technology)

  • You Hee Choi

    (Gwangju Institute of Science and Technology
    Gwangju Institute of Science and Technology)

  • Yunsu Jang

    (Gwangju Institute of Science and Technology
    Gwangju Institute of Science and Technology)

  • Jihyeon Yu

    (Hanyang University
    Hanyang University)

  • Jiyoung Goo

    (Korea Institute of Science and Technology
    Kyunghee University)

  • Gyejun Lee

    (Korea Institute of Science and Technology
    Kyunghee University)

  • You Kyeong Jeong

    (Hanyang University)

  • Seung Hwan Lee

    (Institute for Basic Science
    Korea Research Institute of Bioscience and Biotechnology)

  • In-San Kim

    (Korea Institute of Science and Technology
    Korea University)

  • Jin-Soo Kim

    (Institute for Basic Science
    Seoul National University)

  • Cherlhyun Jeong

    (Korea Institute of Science and Technology
    Kyunghee University)

  • Sanghwa Lee

    (Gwangju Institute of Science and Technology
    Gwangju Institute of Science and Technology)

  • Sangsu Bae

    (Hanyang University
    Hanyang University)

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.

Suggested Citation

  • 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, 2018. "Direct observation of DNA target searching and cleavage by CRISPR-Cas12a," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-05245-x
    DOI: 10.1038/s41467-018-05245-x
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    Cited by:

    1. Margot Karlikow & Evan Amalfitano & Xiaolong Yang & Jennifer Doucet & Abigail Chapman & Peivand Sadat Mousavi & Paige Homme & Polina Sutyrina & Winston Chan & Sofia Lemak & Alexander F. Yakunin & Adam, 2023. "CRISPR-induced DNA reorganization for multiplexed nucleic acid detection," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    2. Santosh R. Rananaware & Emma K. Vesco & Grace M. Shoemaker & Swapnil S. Anekar & Luke Samuel W. Sandoval & Katelyn S. Meister & Nicolas C. Macaluso & Long T. Nguyen & Piyush K. Jain, 2023. "Programmable RNA detection with CRISPR-Cas12a," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    3. Fei Deng & Yi Li & Biyao Yang & Rui Sang & Wei Deng & Maya Kansara & Frank Lin & Subotheni Thavaneswaran & David M. Thomas & Ewa M. Goldys, 2024. "Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    4. Aakash Saha & Mohd Ahsan & Pablo R. Arantes & Michael Schmitz & Christelle Chanez & Martin Jinek & Giulia Palermo, 2024. "An alpha-helical lid guides the target DNA toward catalysis in CRISPR-Cas12a," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

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