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Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information.

Dacheng Ma1, Zhimeng Xu1, Zhaoyu Zhang1

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Summary
This summary is machine-generated.

Researchers engineered chimeric Cas9 variants to expand CRISPR/Cas9 targeting. These new variants recognize diverse PAM sequences, increasing potential genomic targets in mammalian cells.

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biochemistry

Background:

  • CRISPR/Cas9 systems are powerful gene editing tools but are limited by protospacer adjacent motif (PAM) recognition.
  • Staphylococcus aureus Cas9 (SaCas9) has a specific PAM requirement, restricting its targeting scope.

Purpose of the Study:

  • To engineer chimeric Cas9 (cCas9) variants with altered PAM recognition capabilities.
  • To expand the range of genomic sites targetable by CRISPR/Cas9 systems.

Main Methods:

  • Generated chimeric Cas9 variants by swapping the PAM interaction (PI) domain regions of SaCas9 with those from its orthologs.
  • Utilized functional assays to evaluate cCas9 variants at target sites with varied PAM sequences (positions 3-6).

Main Results:

  • Identified several cCas9 variants demonstrating expanded PAM recognition.
  • Observed new recognition capabilities for PAM sequences including NNVRRN, NNVACT, NNVATG, NNVATT, NNVGCT, NNVGTG, and NNVGTT.
  • Engineered variants can access up to 25% of all possible genomic targets in mammalian cells.

Conclusions:

  • Developed a panel of cCas9 variants that significantly broaden CRISPR/Cas9 targeting scope.
  • These engineered nucleases offer enhanced flexibility for gene editing applications in mammalian systems.
  • The expanded PAM recognition expands the utility of CRISPR/Cas9 technology for diverse genomic applications.