Shy Brian R, MacDougall Matthew S, Clarke Ryan, Merrill Bradley J
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA Genome Editing Core, University of Illinois at Chicago, Chicago, IL 60607, USA.
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
Nucleic Acids Res. 2016 Sep 19;44(16):7997-8010. doi: 10.1093/nar/gkw685. Epub 2016 Aug 2.
CRISPR/Cas9 nucleases have enabled powerful, new genome editing capabilities; however, the preponderance of non-homologous end joining (NHEJ) mediated repair events over homology directed repair (HDR) in most cell types limits the ability to engineer precise changes in mammalian genomes. Here, we increase the efficiency of isolating precise HDR-mediated events in mouse embryonic stem (ES) cells by more than 20-fold through the use of co-incidental insertion (COIN) of independent donor DNA sequences. Analysis of on:off-target frequencies at the Lef1 gene revealed that bi-allelic insertion of a PGK-Neo cassette occurred more frequently than expected. Using various selection cassettes targeting multiple loci, we show that the insertion of a selectable marker at one control site frequently coincided with an insertion at an unlinked, independently targeted site, suggesting enrichment of a sub-population of HDR-proficient cells. When individual cell events were tracked using flow cytometry and fluorescent protein markers, individual cells frequently performed either a homology-dependent insertion event or a homology-independent event, but rarely both types of insertions in a single cell. Thus, when HDR-dependent selection donors are used, COIN enriches for HDR-proficient cells among heterogeneous cell populations. When combined with a self-excising selection cassette, COIN provides highly efficient and scarless genome editing.
CRISPR/Cas9核酸酶已具备强大的新型基因组编辑能力；然而，在大多数细胞类型中，非同源末端连接（NHEJ）介导的修复事件比同源定向修复（HDR）更为常见，这限制了在哺乳动物基因组中进行精确编辑的能力。在此，我们通过使用独立供体DNA序列的共插入（COIN），将从小鼠胚胎干细胞（ES细胞）中分离精确的HDR介导事件的效率提高了20多倍。对Lef1基因的靶向频率分析表明，PGK-Neo盒的双等位基因插入比预期更频繁地发生。使用针对多个位点的各种选择盒，我们发现，在一个对照位点插入选择标记常常与在一个不连锁的、独立靶向的位点的插入同时发生，这表明富集了HDR能力强的细胞亚群。当使用流式细胞术和荧光蛋白标记追踪单个细胞事件时，单个细胞通常要么进行同源依赖性插入事件，要么进行同源非依赖性事件，但很少在单个细胞中同时进行这两种类型的插入。因此，当使用依赖HDR的选择供体时，COIN可在异质细胞群体中富集HDR能力强的细胞。当与自我切除的选择盒结合使用时，COIN可提供高效且无疤痕的基因组编辑。
