Genome editing with natural and engineered CjCas9 orthologs.
Mol Ther
; 31(4): 1177-1187, 2023 04 05.
Article
em En
| MEDLINE
| ID: mdl-36733251
ABSTRACT
CjCas9 is one of the smallest CRISPR-associated (Cas9) nucleases for mammalian genome editing. However, it requires a long N4RYAC (R = A or G; Y = C or T) protospacer-adjacent motif (PAM), limiting its DNA targeting scope. In this study, we investigated the PAMs of three CjCas9 orthologs, including Hsp1Cas9, Hsp2Cas9, and CcuCas9, by performing a GFP-activation assay. Interestingly, Hsp1Cas9 and CcuCas9 recognized unique N4RAA and N4CNA PAMs, respectively. We further generated an Hsp1Cas9-Hsp2Cas9 chimeric Cas9 (Hsp1-Hsp2Cas9), which recognized a simple N4CY PAM. Genome-wide off-target analysis revealed that Hsp1-Hsp2Cas9 has very few off-targets compared to SpCas9. By analyzing the crystal structure of CjCas9, we identified eight mutations that can improve the specificity and generate a high-fidelity Hsp1-Hsp2Cas9-Y. Hsp1-Hsp2Cas9-Y enables the knockout of B4GALNT2 and CMAH in porcine fetal fibroblasts (PFFs). Moreover, we developed a high-fidelity Hsp1-Hsp2Cas9-KY which displayed undetectable off-targets revealed by GUIDE-seq at four tested loci. These natural and engineered Cas9 nucleases enabled efficient genome editing in multiple mammalian cells, expanding the DNA targeting scope.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Sistemas CRISPR-Cas
/
Edição de Genes
Limite:
Animals
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article