Bacterial Retrons Enable Precise Gene Editing in Human Cells.
Bacterial Retrons Enable Precise Gene Editing in Human Cells.
复制标题
细菌逆转录子能够在人类细胞中进行精确的基因编辑。
DOI：
10.1089/crispr.2021.0065
复制
发表时间：
2022
期刊：
影响因子：
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通讯作者：
Fraser,HunterB
中科院分区：
文献类型：
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作者：
Zhao,Bin;Chen,Shi-AnA;Lee,Jiwoo;Fraser,HunterB
复制子是参与抗噬菌体防御的细菌遗传元件。它们具有将RNA反向转录成多拷贝单链DNA(MsDNA)的独特能力，该DNA仍与其模板RNA共价连接。在酵母中，复制子与CRISPR-Cas9结合已被证明通过同源定向修复(HDR)提高了精确基因组编辑的效率。在人类细胞中，HDR编辑效率受到与传递编码所需突变的细胞外供体DNA相关挑战的限制。在这项研究中，我们测试了在HEK293T和K562细胞中，通过拴住msDNA来引导RNA，反义寡核苷酸产生msDNA作为供体DNA的能力和促进HDR的能力。通过使用CRISPR-Cas9系统对多个细菌物种的启动子进行异源重组，我们证明了高达11.4%的HDR率。总体而言，我们的发现代表了将基于反转录的精确基因编辑扩展到人类细胞的第一步。
Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve the efficiency of precise genome editing via homology-directed repair (HDR). In human cells, HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.4%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.