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Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e
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Expanding PAM recognition and enhancing base editing activity of Cas9 variants with non-PI domain mutations derived

Lifang Xie1,2, Yun Hu1, Li Li1

  • 1Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Chengdu, China.

The FEBS Journal
|April 12, 2022
PubMed

Insights

Researchers engineered a new CRISPR-Cas9 system, xCas9-NG, by combining protein fragments. This enhanced system broadens the protospacer adjacent motif recognition, improving genome editing efficiency and targeting scope.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The CRISPR-Cas9 system's targeting range is limited by its recognition of the protospacer adjacent motif (PAM).
  • The PAM interacting (PI) domain of Cas9 is crucial for PAM specificity, but other regions may also influence recognition.
  • Understanding these interactions is key to expanding CRISPR-Cas9 applications.

Purpose of the Study:

  • To engineer a novel Cas9 variant with expanded PAM recognition.
  • To investigate the role of non-PI domains in PAM interaction and specificity.
  • To improve the genome editing efficiency and targeting scope of CRISPR-Cas9 systems.

Main Methods:

  • Constructed a mosaic SpCas9 protein (xCas9-NG) by fusing the PI domain of Cas9-NG with a modified non-PI fragment of xCas9.
  • Assessed the PAM recognition profile of the engineered xCas9-NG variant.
  • Evaluated the genome editing efficiency of xCas9-NG across various targets.

Main Results:

  • The non-PI fragment of xCas9 successfully expanded the PAM recognition of the Cas9-NG PI domain.
  • xCas9-NG demonstrated improved editing efficiency for targets with xCas9 and Cas9-NG PAMs.
  • The findings were validated in other Cas9 variants, including SpRY and non-G SpCas9 series.

Conclusions:

  • The non-PI domain fragment significantly contributes to PAM restriction and recognition specificity.
  • The engineered xCas9-NG expands the targeting scope of the SpCas9 editing system.
  • This work provides a foundation for developing more versatile CRISPR-Cas9 tools for genome editing.
Keywords:
CRISPR-Cas9Cas9 variantsPAMbase editingxCas9

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