A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing / 生物工程学报
Chinese Journal of Biotechnology
; (12): 4204-4218, 2023.
Article
em Zh
| WPRIM
| ID: wpr-1008021
Biblioteca responsável:
WPRO
ABSTRACT
During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.
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Índice:
WPRIM
Assunto principal:
DNA
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Células HEK293
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Recombinação Homóloga
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Sistemas CRISPR-Cas
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Edição de Genes
Limite:
Animals
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Humans
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2023
Tipo de documento:
Article