Exonuclease editor promotes precision of gene editing in mammalian cells

• Published in: BMC biology 2024-05, Vol.22 (1), p.119-119, Article 119
• Main Authors: Shi, Hui, Li, Lei, Mu, Shuangshuang, Gou, Shixue, Liu, Xiaoyi, Chen, Fangbing, Chen, Menglong, Jin, Qin, Lai, Liangxue, Wang, Kepin
• Format: Article
• Language: English
• Subjects: Cell cycle; CRISPR-Cas Systems; DMD; DNA damage; DNA repair; DNA Repair Enzymes; Editing; Editors; Efficiency; Exodeoxyribonucleases - genetics; Exodeoxyribonucleases - metabolism; Exonuclease; Exonuclease editor; Exonucleases; Gene correction; Gene deletion; Gene Editing - methods; Gene therapy; Genes; Genetic aspects; Genetic modification; Genome editing; Genomes; Health aspects; HEK293 Cells; Homology-directed repair; Humans; Kinases; Mammalian cells; Mammals; Medical research; Medicine, Experimental; Microhomology-based precise deletion; Non-homologous end joining; Peptides; Pluripotency; Proteins; Stem cells
• ISSN: 1741-7007; 1741-7007
• DOI: 10.1186/s12915-024-01918-w

Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.