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Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease

Heng Zhang1, Zhuang Li2, Renjian Xiao2

  • 1Department of Biological Sciences, Purdue University, West Lafayette, IN, USA. zhangheng134@gmail.com.

Nature Structural & Molecular Biology
|September 8, 2020
PubMed

Insights

Cas12i, a CRISPR-Cas enzyme, shows potential for precise genome editing by nicking DNA. Its mechanism involves specific RNA-DNA binding, enabling targeted DNA modification for advanced gene editing applications.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • CRISPR-Cas systems are powerful tools for genome engineering.
  • Cas12i, a Type V CRISPR-Cas endonuclease, exhibits unique DNA nicking activity.
  • Understanding Cas12i's mechanism is crucial for its application in precise genome editing.

Purpose of the Study:

  • To elucidate the target recognition and DNA cleavage mechanisms of Cas12i.
  • To determine the cryo-electron microscopy (cryo-EM) structures of Cas12i in various functional states.
  • To provide mechanistic insights for optimizing Cas12i in genome editing.

Main Methods:

  • Cryo-electron microscopy (cryo-EM) to determine high-resolution structures.
  • Biochemical assays to study DNA cleavage activity.
  • Analysis of crRNA-DNA hybridization and base pairing interactions.

Main Results:

  • Cas12i utilizes a seven-nucleotide seed sequence for target recognition.
  • A two-step activation process is mediated by crRNA-DNA hybridization.
  • Nickase activity is activated upon formation of 14 base pairs, and target strand cleavage occurs at 28 base pairs.

Conclusions:

  • The determined structures reveal the mechanistic basis of Cas12i's specific DNA targeting and cleavage.
  • Cas12i's unique nicking activity and stepwise activation offer potential for highly specific genome editing.
  • These findings pave the way for the rational design and application of Cas12i in advanced gene editing technologies.

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