| CRISPR-Cas9 gene editing mainly depends on DNA targeted cleavage and repair.Different repair pathways end with different repair results.Therefore,exploring the repair pathway and corresponding pathway elements after Cas9 targeted cleavage can help to achieve more precise gene editing though manipulate the repair pathway choice;Gene editing pigs are of great significance in biomedicine as disease models or organ transplant donors.However,due to species differences,the regulation of many known repair pathway elements is significantly different in pig cells.Therefore,this paper applies high-throughput CRISPR-Cas9 gene editing technology to explore the Cas9 cleavage repair pathway and key regulatory elements in pig cells at the genome-wide level,laying the foundation for more accurate gene editing.In this paper,about 114095 sgRNAs were designed for 20338 genes of the pig genome.A porcine genome-wide CRISPR-Cas9 virus library was constructed via CRISPR-Cas9 system.The genome-wide knockout cell library was obtained by infecting PFF cells at a MOI of 0.3,and library quality control was performed at different time points to test library stability.At the same time,this paper designs and constructs two Cas9-mediated DSB repair reporter systems:traditional promoterless homologous recombination repair reporter system: pUC57-GAPDHEGFP-Reporter,which can reflect target-specific homologous recombination repair outcomes by green fluorescence;and a new reporter system based on high-throughput sequencing of target repair.In this system,the sequence near the library sgRNA was chosen as the CRISPRCas9 target,and target repair results with library sgRNA were obtained at the same time via the amplicon sequencing.Finally,based on the differences in the abundance of sgRNAs in different repair outcomes after Cas9 cleavage,key specific genes for different repair pathways were screened.The research results were as follows:(1)The sgRNA amplicon sequencing of the constructed pig CRISPR-Cas9 whole-genome knockout cell library at 24 h and 18-day showed that both reads mapping ratio was higher than 65%,and the sgRNA presentation rate was higher than 99.5%.The Gini coefficient of library sgRNA distribution is less than 0.2,which are all meet the library quality control standards,indicating that the Cas9 whole-genome knockout PFF library has been successfully constructed;(2)When validating the promoterless HR repair reporter system,EGFP leakage expression was observed in control groups of PFF and PK15cells(transfected with reporter plasmid only),whom genome was not cut by CRISPR-Cas9.Furthermore,this leaky expression was also observed in cells of other species,such as 293 T and CHO-K1 cells,up to 49% green fluorescence was even observed in HepaRG,indicating that the specificity of this traditional reporter system is not good.In contrast,the novel reporter system based on NGS of target repair is more accurate and intuitive to classify by sequencing results.(3)The target repair-seq results were then classified into four repair types according to Scar Mapper.At the same time,the sgRNA sequence coupled with the repair results was extracted,and the corresponding sgRNA abundance differences in different repair types were analyzed by comparing with non-target sgRNA group.RAD50,DMAP1,PIK3R2 etc.were enriched in Insertion repair results;ZBTB1,NOX4,LBX2 etc.were enriched in Non-MH Deletion repair results;NRDC、KLHL42 etc.were enriched in TMEJ repair results;Genes such as ARIH2 and GOLM1 were enriched NHEJ repair results,suggesting these genes might facilitate the corresponding repair outcomes.In conclusion,this study successfully established a porcine CRISPR-Cas9 genome-wide knockout library and based on this cell library combined with a novel repair outcome reporter system,a series of porcine Cas9 cleavage repair related genes were found,laying the molecular foundation for further realization of more precise porcine Cas9 gene editing. |
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