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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

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A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

Peng Chen1, Jin Zhou1, Yibin Wan1

  • 1State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China.

Genome Biology
|March 28, 2020
PubMed
Abstract

Insights

A new Cas12a enzyme, CeCas12a, offers precise genome editing in human cells. It exhibits high efficiency and reduced off-target edits, especially at non-canonical PAM sites, making it a promising tool for gene editing applications.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Cas12a nucleases (AsCas12a, LbCas12a) are key genome engineering tools.
  • Optimal protospacer adjacent motif (PAM) is TTTV, but C-containing PAMs cause off-target edits.

Purpose of the Study:

  • Identify and characterize a novel Cas12a nuclease with improved specificity.
  • Evaluate CeCas12a's genome editing efficiency and off-target profile in human cells.

Main Methods:

  • Identification of CeCas12a from Coprococcus eutactus.
  • Assessed genome editing efficiency and PAM recognition stringency in vitro and in vivo.
  • Utilized targeted sequencing to evaluate off-target edits at canonical and non-canonical PAM sites.

Main Results:

  • CeCas12a demonstrates genome editing efficiencies comparable to AsCas12a and LbCas12a in human cells.
  • CeCas12a exhibits stringent PAM recognition, leading to significantly lower off-target edits.
  • Reduced off-target edits were observed at C-containing PAM sites compared to LbCas12a and AsCas12a.

Conclusions:

  • CeCas12a is an active nuclease in human cells with enhanced specificity.
  • PAM recognition stringency is crucial for minimizing off-target edits in gene editing.
  • CeCas12a presents a valuable tool for research and therapeutic gene editing applications.
Keywords:
CRISPRCas12aGene editingOff-targetingPAM stringency

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