Development And Application Of Paired-sgRNAs And Cas9-mediated Cell Enrichment System For Precisely Deletion

The precise genome editing is becoming an important tool for studying gene function,treating diseases and developing new animal species as it can safely alter genomes accurately.The development of the CRISPR/Cas9 system has made it easier and more efficient for scientists to edit genomes.In the CRISPR/Cas9 system,Cas9 nuclease was guided by single-stranded guiding RNA(sg RNA)and cleave the sequence of the target genomic site,causing DNA double-strand breaks(DSBs).There are two main ways to repair DNA double strands break: non-homologous end connection(NHEJ)and homologous recombination(HDR).Classical NHEJ is considered to be an error-prone DSBs repair pathway,which usually results in random insertion or deletion(indels)by the target sites,and its efficiency is much higher than that of HDR.The latter is more widely used in the field of precise genome editing,such as base pair replacement,gene fragment insertion and deletion.Recent studies have found that there is a special NHEJ repair pattern.When Cas9 nuclease leads to the repair of double strands break of DNA,the two dissociative DNA ends are not digested and modified,but directly connected back by DNA ligase.This special repairment is known as Accurate-NHEJ(ac NHEJ).Unlike the classic NHEJ pathway,ac NHEJ repair is precise and predictable.Researchers found that ac NHEJ was almost as efficient as the classic NHEJ.However,the reported length of ac NHEJ-mediated accurate segment deletion was only within 100 bp,and there was no reported about ac NHEJ deletion for long segments.In order to verify that the ac NHEJ repairment can mediate the precise deletion of long fragments,and applied it to the study of precise deletion of genes induced by CRISPR/Cas9.In this study,we developed a universal surrogate reporter system for enrichment of precise deletion of cells based on ac NHEJ repair mediated by pairs sg RNA and Cas9,which we dubbed “ac NHEJ-USR”(Accurate NHEJ-based Universal Surrogate Reporter).The reporter system has the characteristics of self-cutting and self-repairment.The reporter vector was co-transferred to the cell with the paired sg RNA targeted vector,and then the cells were screened by purinomycin for 5 days,and the ac NHEJ-mediated precise deletion of cells could be enriched efficiently.This system can be widely used in the study of gene deletion in mammalian cells.The main results of this study are shown as follows:1.We constructed the ac NHEJ-USR vector,which includes two universal sg RNA expression cassette,one disrupted purinomycin resistance gene with universal sg RNA target site,and one green fluorescent protein expression cassette in series.After co-transfection of ac NHEJ-USR and targeting vectors into cells,the paired sg RNAs which were expressed by gene targeting vectors lead Cas9 nuclease cutting the two loci within the puromycin gene,then the reporter vector repaired in the form of ac NHEJ,resulting in the puromycin gene expression.2.Using ac NHEJ-USR enrichment system to precise deletion of the AAVS1,EMX1 and CCR5 gene loci in HEK293 T cells.The screened cells' genome DNA was analysis by PCR and sequencing of target sequence.The long segment deletion efficiency of the three genes after ac NHEJ-USR enrichment was 91.69%,90.63% and 84.38%,respectively,which was 3.0 times,3.3 times and 3.9 times higher than that of the unscreened group.At the same time,the precise deletion efficiency of the long fragments of the three genes enriched by ac NHEJ-USR was 82.29%,70.84% and 63.55%,which was 6.6 times,6.2 times and 7.6 times higher than that of the unscreened group.3.Using the ac NHEJ-USR enrichment system to precise deletion lnc-sscg3623 in porcine PK15 cells.After the significant expression of lnc-sscg3623 in pig adipose tissue was determined,PK15 cells were co-transfected by the ac NHEJ-USR vector and the paired sg RNA target vectors targeting the lnc-sscg3623 genome loci.The cells pool genome DNA was identified by PCR and analyzed by sequencing.Lnc-sscg3623 was precisely bi-allelic deleted with efficiency of 10%.4.High-throughput sequencing analysis was performed on cell clones of homozygous precisely deleted by lnc-sscg3623.Twenty candidate genes with significant expression changes which related to fat metabolism were screened out,and the reliability of the sequencing results was verified by q RT-PCR.After the overexpression of lnc-sscg3623,it was found that only the expression of ACAA2 gene was in reverse synchronization with the expression of lnc-sscg3623 within the candidate genes.Combined with the nuclear and cytoplasmic separation and differentiation induction of porcine adipocyte,it was found that lnc-sscg3623 inhibited porcine adipocyte differentiation by negatively regulating the expression of ACAA2 gene.The precise deletion of lnc-sscg3623 in pigs proved that the ac NHEJ-USR system can be used to study the precise deletion of non-coding RNA.In conclusion,the ac NHEJ-USR system can be used to screen and enrich precisely deleted cells in a simple and efficient manner without affecting the integrity of the genome.Finally,this reporter system can enable the accurate deletion of long segments of genes mediated by paired sg RNAs and Cas9 based on ac NHEJ repairment to be more widely used in the study of gene function,the development of new species of animals and the establishment of gene deletion model animals.