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Related Experiment Video

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Efficient genome editing by CRISPR-Mb3Cas12a in mice.

Zhuqing Wang1, Yue Wang1, Shawn Wang1

  • 1Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV 89557, USA.

Journal of Cell Science
|May 13, 2020
PubMed
Summary

This study introduces Mb3Cas12a for efficient genome editing in mice, utilizing the TTV PAM sequence. It achieves high editing and knock-in efficiencies in mouse embryos, offering a valuable alternative to Cas9.

Keywords:
CRISPRCas12aCas9Cpf1Genome editingPAM

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Cas12a is an alternative to Cas9 for genome editing but has limited use in mammalian cells due to its strict TTTV protospacer adjacent motif (PAM) requirement.
  • Efficient genome editing tools are crucial for genetic research and therapeutic development.

Purpose of the Study:

  • To investigate the efficiency of Mb3Cas12a from *Moraxella bovoculi* for genome editing in mammalian systems.
  • To assess the utility of Mb3Cas12a with a TTV PAM sequence for gene knock-in and editing in mouse embryos.

Main Methods:

  • Utilized Mb3Cas12a with 23 nt CRISPR (cr)RNAs targeting the TTV PAM sequence for genome editing in mouse.
  • Employed Mb3Cas12a fused to monomeric streptavidin (mSA) with biotinylated DNA donor templates for knock-in experiments in two-cell mouse embryos.

Main Results:

  • Mb3Cas12a demonstrated efficient editing of the mouse genome with minimal large on-target deletions or insertions.
  • >70% of founders obtained showed successful editing when using Mb3Cas12a with TTTV PAM-targeting crRNAs.
  • High knock-in efficiency (40% of founders) was achieved in two-cell mouse embryos using mSA-Mb3Cas12a and biotinylated DNA donors.

Conclusions:

  • Mb3Cas12a is an effective tool for genome editing in mammalian cells, overcoming the limitations of other Cas12a variants.
  • The TTV PAM specificity of Mb3Cas12a and its compatibility with mSA-tagging and biotinylated donors enhance its applicability for precise genome engineering in mouse models.