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Highly efficient base editing with expanded targeting scope using SpCas9-NG in rabbits.

Zhiquan Liu1, Huanhuan Shan1, Siyu Chen1

  • 1Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun, China.

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
|January 10, 2020
PubMed
Summary

Engineered base editors (NG-BE4max and NG-ABEmax) efficiently create precise point mutations in rabbit embryos by recognizing NG PAMs. These tools expand genome editing capabilities for modeling human diseases in rabbits.

Keywords:
CRISPR/Cas9NG PAMsbase editorrabbit

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Area of Science:

  • Molecular Biology
  • Genome Editing
  • Animal Models

Background:

  • Base editors enable precise DNA base conversions (C-to-T or A-to-G) but are limited by the protospacer adjacent motif (PAM) recognition of Streptococcus pyogenes Cas9 (SpCas9).
  • Engineered SpCas9-NG variants offer expanded PAM compatibility, recognizing minimal NG PAMs, which broadens targetable sites for base editing.

Purpose of the Study:

  • To evaluate the efficiency and PAM compatibility of SpCas9-NG-assisted cytidine base editors (CBEs) and adenine base editors (ABEs) in rabbit embryos.
  • To demonstrate the utility of these enhanced base editors in generating precise point mutations for creating novel rabbit models.

Main Methods:

  • Utilized NG-BE4max and NG-ABEmax systems in rabbit embryos.
  • Assessed targeted mutation efficiency and PAM compatibility across various NGN PAM sites.
  • Applied base editors to generate specific point mutations, including mimicking a human missense mutation.

Main Results:

  • NG-BE4max achieved 75%-100% targeted mutation efficiency with NGN PAM compatibility in rabbit embryos.
  • NG-ABEmax demonstrated 80%-100% targeted mutation efficiency with NGN PAM compatibility.
  • Successfully generated rabbit models with precise point mutations, showcasing high efficiency and expanded targeting scope.
  • NG-ABEmax precisely mimicked the human Hoxc13 p.Q271R missense mutation, overcoming limitations of conventional ABEs.

Conclusions:

  • NG-BE4max and NG-ABEmax systems are highly efficient tools for base editing in rabbits.
  • These systems offer expanded genome-targeting scope due to their NG PAM compatibility.
  • They enhance the capacity for creating rabbit models to study human diseases.