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

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.

SUBMITTER: Ma D

PROVIDER: S-EPMC6361995 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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

Ma Dacheng D Xu Zhimeng Z Zhang Zhaoyu Z Chen Xi X Zeng Xiangzhi X Zhang Yiyang Y Deng Tingyue T Ren Mengfei M Sun Zheng Z Jiang Rui R Xie Zhen Z

Nature communications 20190204 1

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 wit ...[more]

PMID: 30718489

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