Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Biomolecules. 2022 Nov 3;12(11):1621. doi: 10.3390/biom12111621.
In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in . The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing.
在精确的基因组编辑中，CRISPR/Cas9 系统诱导的靶部位 DNA 双链断裂（DSBs）通过同源定向修复（HDR）利用外源供体 DNA 模板进行修复。然而，HDR 介导的基因组编辑效率低是广泛应用的障碍。在本研究中，我们构建了一种供体 DNA/guide RNA（gRNA）杂交双链体（DGybrid），它由序列延伸的 gRNA 和单链寡脱氧核苷酸（ssODN）组成，结合互补碱基，而不进行化学修饰，以增加切割部位供体 DNA 的浓度。我们在. 中评估了使用 DGybrid 进行基因组编辑的效率。结果表明，与单独导入 gRNA 和供体 DNA 的基因组编辑相比，HDR 介导的编辑效率提高了 1.8 倍（从 35%提高到 62%）。此外，通过流式细胞术分析核酸导入效率表明，通过使用 DNA/RNA 杂交，RNA 和 ssODN 可以一起有效地共同被细胞摄取。我们的技术将作为一种通用且简洁的工具，用于提高 HDR 介导的基因组编辑效率。
