通过阵列式标签高通量测序高效鉴定点突变单克隆细胞

Efficient identification of point-mutant monoclonal cells through array tagged high-throughput sequencing

目的建立阵列式标签标记的高通量测序方法并结合多克隆预筛选实现对点突变单克隆细胞株的高效鉴定。方法通过CRISPR/Cas9介导的同源重组修复(HDR)在K562细胞的rs826415位点引入G到T点突变。侯选细胞按每孔10~20个的密度接种培养多克隆,以带有标签(barcode)的引物区分各多克隆细胞,PCR扩增含rs826415位点的区段,产物合并进行二代测序。生物信息学分析各多克隆细胞的同源重组率及插入/缺失率。将阳性率最高孔内的细胞进一步单克隆培养并鉴定基因型。结果通过CRISPR/Cas9同源重组法和有限稀释获得96个rs826415位点打靶的多克隆细胞。经阵列式标签高通量测序法分析各孔细胞同源重组且不伴随插入/缺失(HDR without indel)的比率,96个多克隆的平均阳性率为0.21%,最高达4.35%,将该多克隆细胞进一步单克隆培养,从30个单克隆中成功获得rs826415位点由G/G突变为T/G的杂合型细胞株。结论通过阵列式标签高通量测序策略结合多克隆细胞预筛选,实现了定点突变单克隆细胞株的高效鉴定,相比常规方法显著降低了工作量和成本。

Objective To establish an array tagged high-throughput sequencing and pre-polyclonal screening strategy for fast identification of genome-edited cell clones.Methods Introducing of G to T point mutation at rs826415 site of K562 cells by CRISPR/Cas9-mediated homologous recombination.The candidate cells were then seeded by 10~20 cells per well in a 96-well-plateand and so to be expanded into 96 polyclones.A two-step nested PCR with array barcoded primers was used to amplify the rs826415 surrounding region to differentially tag PCR product from each polyclone.The PCR products were mixed and subjected to next-generation sequencing to detect the recombination and indel rate for each polyclone.The polyclonal cells carrying the highest‘homology-directed repair(HDR)without indel’rate were sorted for monoclonal growth and rs826415 genotyping.Results Polyclonal cells obtained by CRISPR/Cas9-mediated homologous recombination and limiting dilution were screened with array tagged high-throughput sequencing.Ratio of the cells showed‘HDR without indel’was determined for each polyclone.Of the 96 polyclones screened,an average‘HDR without indel’rate was 0.21%,and the highest rate was 4.35%.Cells in the later were then subjected to monoclonal growth in a new 96-well-plate.In the 30 monoclones obtained,one showed the expected hybrid T/G genotype at the rs826415 site,instead of the G/G genotype in the unmodified K562 cells,indicated successful mono-allele targeting.Conclusions The array tagged high-throughput sequencing strategy,in combination with a pre-polyclonal screening,provides an efficient method for fast identification of genome-edited monoclonal cell.It saves both human and financial resources as compares to conventional screening methods.