A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing.

Baoxia MA; Jieyu CUI; Hongrun QIAN; Xiaojun ZHANG; Sen YANG; Qijing ZHANG; Yifan HAN; Zhiying ZHANG; Jiangang WANG; Kun XU

Return

A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing.

Author: Baoxia MA ¹ ; Jieyu CUI ¹ ; Hongrun QIAN ¹ ; Xiaojun ZHANG ¹ ; Sen YANG ¹ ; Qijing ZHANG ¹ ; Yifan HAN ¹ ; Zhiying ZHANG ¹ ; Jiangang WANG ¹ ; Kun XU ¹
Author Information

1. College of Animal Science and Technology, Northwest A & F University, Yangling 712100, Shaanxi, China.
Publication Type:Journal Article
Keywords: CRISPR/Cas9; LacI gene; LacO sequence; donor adapting; gene editing
MeSH: Humans; Animals; Gene Editing; CRISPR-Cas Systems/genetics*; HEK293 Cells; Homologous Recombination; DNA
From: Chinese Journal of Biotechnology 2023;39(10):4204-4218
CountryChina
Language:Chinese
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.