首页|Detailed mechanisms for unintended large DNA deletions with CRISPR, base editors, and prime editors
Detailed mechanisms for unintended large DNA deletions with CRISPR, base editors, and prime editors
Detailed mechanisms for unintended large DNA deletions with CRISPR, base editors, and prime editors
基础医学生物科学研究方法、生物科学研究技术遗传学
Oh Minsik,Kim Segi,Habib Omer,Kim Chan Hyuk,Kim Sun,Kim Seok-Hoon,Hwang Gue-ho,Bae Sangsu,Jang Hyeon-Ki,Kim Heon Seok.Detailed mechanisms for unintended large DNA deletions with CRISPR, base editors, and prime editors[EB/OL].(2025-03-28)[2025-09-24].https://www.biorxiv.org/content/10.1101/2024.01.04.574288.点此复制
CRISPR-Cas9 nucleases are versatile tools for genetic engineering cells and function by producing targeted double-strand breaks (DSBs) in the DNA sequence. However, the unintended production of large deletions (>100 bp) represents a challenge to the effective application of this genome-editing system. We optimized a long-range amplicon sequencing system and developed a k-mer sequence-alignment algorithm to simultaneously detect small DNA alteration events and large DNA deletions. With this workflow, we determined that CRISPR-Cas9 induced large deletions at varying frequencies in cancer cell lines, stem cells, and primary T cells. With CRISPR interference screening, we determined that end resection and the subsequent TMEJ [DNA polymerase theta-mediated end joining] repair process produce most large deletions. Furthermore, base editors and prime editors also generated large deletions despite employing mutated Cas9 nickases that produce single-strand breaks. Our findings reveal an important limitation of current genome-editing tools and identify strategies for mitigating unwanted large deletion events.
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