Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.
Section of Molecular Biology, University of California San Diego, La Jolla, CA, USA.
Nat Commun. 2021 May 11;12(1):2625. doi: 10.1038/s41467-021-22927-1.
CRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.
基于 CRISPR 的活性遗传元件（或基因驱动）通过同源定向修复（HDR）在生殖系中复制，以超孟德尔频率传递给后代。活性遗传元件还可以产生广泛的体细胞突变，但这种表型的遗传基础尚不确定。通常认为，这种体细胞突变是由非同源末端连接（NHEJ）产生的，NHEJ 是体细胞中活性最强的双链断裂修复途径。在这里，我们在果蝇中开发了 CopyCatcher 系统来检测和量化体细胞基因转换（SGC）事件。插入到两个独立遗传位点的 CopyCatchers 揭示了果蝇眼睛和胸部表皮中出人意料的高 SGC 率。基于 RNAi 的聚焦遗传筛选确定了几个改变 SGC 效率的意外基因座，其中一个（c-MYC）下调时，会促进人 HEK293T 细胞中质粒和同源染色体模板介导的 SGC。总的来说，这些研究表明，CopyCatchers 可以作为有效的发现平台，为潜在的基因治疗策略提供信息。
