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Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information

Author

Listed:
  • Dacheng Ma

    (Tsinghua University)

  • Zhimeng Xu

    (Tsinghua University)

  • Zhaoyu Zhang

    (Tsinghua University)

  • Xi Chen

    (Tsinghua University)

  • Xiangzhi Zeng

    (Tsinghua University)

  • Yiyang Zhang

    (Tsinghua University)

  • Tingyue Deng

    (Tsinghua University)

  • Mengfei Ren

    (Tsinghua University)

  • Zheng Sun

    (Tsinghua University)

  • Rui Jiang

    (Tsinghua University)

  • Zhen Xie

    (Tsinghua University)

Abstract

Although Cas9 nucleases are remarkably diverse in microorganisms, the range of genomic sequences targetable by a CRISPR/Cas9 system is restricted by the requirement of a short protospacer adjacent motif (PAM) at the target site. Here, we generate a group of chimeric Cas9 (cCas9) variants by replacing the key region in the PAM interaction (PI) domain of Staphylococcus aureus Cas9 (SaCas9) with the corresponding region in a panel of SaCas9 orthologs. By using a functional assay at target sites with different nucleotide recombinations at PAM position 3–6, we identify several cCas9 variants with expanded recognition capability at NNVRRN, NNVACT, NNVATG, NNVATT, NNVGCT, NNVGTG, and NNVGTT PAM sequences. In summary, we provide a panel of cCas9 variants accessible up to 1/4 of all the possible genomic targets in mammalian cells.

Suggested Citation

  • Dacheng Ma & Zhimeng Xu & Zhaoyu Zhang & Xi Chen & Xiangzhi Zeng & Yiyang Zhang & Tingyue Deng & Mengfei Ren & Zheng Sun & Rui Jiang & Zhen Xie, 2019. "Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information," Nature Communications, Nature, vol. 10(1), pages 1-9, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-08395-8
    DOI: 10.1038/s41467-019-08395-8
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    Cited by:

    1. Dawn G. L. Thean & Hoi Yee Chu & John H. C. Fong & Becky K. C. Chan & Peng Zhou & Cynthia C. S. Kwok & Yee Man Chan & Silvia Y. L. Mak & Gigi C. G. Choi & Joshua W. K. Ho & Zongli Zheng & Alan S. L. W, 2022. "Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    2. Lin Zhao & Sabrina R. T. Koseki & Rachel A. Silverstein & Nadia Amrani & Christina Peng & Christian Kramme & Natasha Savic & Martin Pacesa & Tomás C. Rodríguez & Teodora Stan & Emma Tysinger & Lauren , 2023. "PAM-flexible genome editing with an engineered chimeric Cas9," Nature Communications, Nature, vol. 14(1), pages 1-8, December.

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