Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

Bo Zhang, Diyin Luo, Yu Li, Vanja Perčulija, Jing Chen, Jinying Lin, Yangmiao Ye and Songying Ouyang ()
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Bo Zhang: Fujian Normal University
Diyin Luo: Fujian Normal University
Yu Li: Fujian Normal University
Vanja Perčulija: Fujian Normal University
Jing Chen: Fujian Normal University
Jinying Lin: Fujian Normal University
Yangmiao Ye: Fujian Normal University
Songying Ouyang: Fujian Normal University

Nature Communications, 2021, vol. 12, issue 1, 1-13

Abstract: Abstract Cas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.

Date: 2021
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DOI: 10.1038/s41467-021-23876-5

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