CRISPR/Cas9-mediated homology-directed repair (HDR) can be leveraged to precisely engineer mammalian genomes. However, the inherently low efficiency of HDR often hampers to identify the desired modified cells. Here, we developed a novel universal surrogate reporter system that efficiently enriches for genetically modified cells arising from CRISPR/Cas9-induced HDR events (namely, the “HDR-USR” system). This episomally based reporter can be self-cleaved and self-repaired via HDR to create a functional puromycin selection cassette without compromising genome integrity. Co-transfection of the HDR-USR system into host cells and transient puromycin selection efficiently achieves enrichment of HDR-modified cells. We tested the system for precision point mutation at 16 loci in different human cell lines and one locus in two rodent cell lines. This system exhibited dramatic improvements in HDR efficiency at a single locus (up to 20.7-fold) and two loci at once (42% editing efficiency compared to zero in the control), as well as greatly improved knockin efficiency (8.9-fold) and biallelic deletion (35.9-fold) at test loci. Further increases were achieved by co-expression of yeast Rad52 and linear single-/double-stranded DNA donors. Taken together, our HDR-USR system provides a simple, robust and efficient surrogate reporter for the enrichment of CRISPR/Cas9-induced HDR-based precision genome editing across various targeting loci in different cell lines.

CRISPR / Cas9介导的同源性定向修复（HDR）可用于精确改造哺乳动物基因组。但是，HDR固有的低效率通常会妨碍识别所需的修饰细胞。在这里，我们开发了一种新型的通用替代报告系统，可有效富集由CRISPR / Cas9诱导的HDR事件产生的基因修饰细胞（即“ HDR-USR”系统）。该基于情境的报道分子可以通过HDR自我切割和自我修复，以创建功能性的嘌呤霉素选择盒，而不会损害基因组完整性。将HDR-USR系统共转染到宿主细胞中，并进行短暂的嘌呤霉素选择，可以有效地实现HDR修饰细胞的富集。我们测试了该系统在不同人类细胞系中16个基因座和两个啮齿动物细胞系中一个基因座的精确点突变。该系统在单个基因座（高达20.7倍）和同时两个基因座（编辑效率为42％，而对照组为零）中显着提高了HDR效率，并极大地提高了敲入效率（8.9倍）和测试位点的双等位基因缺失（35.9倍）。通过酵母Rad52和线性单链/双链DNA供体的共表达实现了进一步的增加。综上所述，我们的HDR-USR系统提供了一个简单，强大且高效的替代报告基因，可丰富CRISPR / Cas9诱导的基于HDR的精确基因组编辑，可跨越不同细胞系中的各个靶向基因座。9倍）和双等位基因缺失（35.9倍）。通过酵母Rad52和线性单链/双链DNA供体的共表达实现了进一步的增加。综上所述，我们的HDR-USR系统提供了一个简单，强大且高效的替代报告基因，可丰富CRISPR / Cas9诱导的基于HDR的精确基因组编辑，可跨不同细胞系中的各个靶向基因座进行编辑。9倍）和双等位基因缺失（35.9倍）。通过酵母Rad52和线性单链/双链DNA供体的共表达实现了进一步的增加。综上所述，我们的HDR-USR系统提供了一个简单，强大且高效的替代报告基因，可丰富CRISPR / Cas9诱导的基于HDR的精确基因组编辑，可跨不同细胞系中的各个靶向基因座进行编辑。