关键词: CRISPR/Cas9, 同源重组, 阵列式标签高通量测序, 克隆筛选

Abstract: 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.

Key words: CRISPR/Cas9, homologous recombination, array tagged high-throughput sequencing, clone screening