Work

High-Frequency Generation of Homozygous/Biallelic Mutants via CRISPR/Cas9 Driven by AtKu70/80 Promoters

Year: 2025

Type: article

Abstract: CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, Cas9 expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving Cas9 with promoters of AtKu70 and AtKu80, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of AtKu70 and AtKu80 were cloned and used to drive Cas9 in the CRISPR/Cas9 system. Four different genes, GmRj7, GmNNL1, AtPDS3, and AtBRI1, were designed for soybean hairy root transformation and Arabidopsis transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of AtKu70 and AtKu80 achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by Rhizobium rhizogenes and stable genetic transformation with Rhizobium tumefaciens.

Source: International Journal of Molecular Sciences

DOI: 10.3390/ijms26189094

Authors: Huihui Zhang, Chong Teng, Shanhua Lyu, Yinglun Fan

Institution: Liaocheng University

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,499

Funder: Natural Science Foundation of Shandong Province