CRISPR/Cas9 Mediated Generation Of Stable Cell Lines With Editing Drosha, XRCC6, RNase L Genes

In recent years, the development of gene editing techniques, including zinc finger nucleases(ZFNs), transcription activator-like type II effector nucleases(TALENs) and clustered regularly interspaced short palindromic repeats(CRISPRs)/Cas9 system allow scientists to more rapidly and efficiently edit the genome and study the role of genes.CRISPR/Cas system as a kind of the newest gene editing tools, can efficiently and selectively edit genome and change the function of genes in vivo and in vitro and will greatly promote biological and biomedical research. In this paper, we applied a CRISPR/Cas9 system for genome editing of HEK293 cells and respectively disrupted Drosha gene and RNase L gene, for genome editing of HeLa cells and inserted Flag tag at the C-terminal of XRCC6 gene.We firstly obtained HEK293 cell lines with disruption of Drosha gene using CRISPR/Cas9 system. The hygromycin B resistance gene sequence with the termination codon was inserted into Drosha gene and the inserted site near the initiation codon of Drosha in order to mutate the open reading frame of Drosha, and the insertion of hygromycin B resistance gene would improve the screening efficiency of obtaining cell lines with disruption of Drosha gene. According to the sequence of Drosha in the GenBank, small guide RNA(sgRNA) sequences of human Drosha were designed and sgRNAs were inserted into pCas-Guide and obtained pCas-sgRNA vectors. Then we designed the donor DNA sequences of homologous arm for disruption of Drosha gene and in the presence of right homologous arm, the resistance gene of hygromycin B and left homologous arm as templates of homology-directed repair. The donor DNAtemplate was amplified through overlopping PCR and cloned into the pBackZero-T expression vector and obtained pBackZero-T-Drosha vector. The pCas-sgRNA vector and pBackZero-T- Drosha vector were co-transfected into HEK293 cells to obtain the stable expression cell line of disruption of Drosha gene. Culturing cells with hygromycin B, western blot and DNA sequencing were used to analyze disruption of Drosha gene from genome. Using the same method, we also obtained five HEK293 cell lines with disruption of RNase L gene and studied the effects of RNase L on cell proliferation, cell cycle and cell apoptosis.In addition to gene knockout, we also obtained XRCC6-Flag cell lines that Flag tag was inserted at the C-terminus of XRCC6 gene in the genome by CRISPR/Cas9 system.According to the sequence of XRCC6 in the GenBank, sgRNA sequences were designed and inserted into the pCas-Guide vectors and pCas-XRCC6 vectors were obtained. The sequences of homologous arms of XRCC6 gene were designed. Donor DNA were amplified by PCR with homologous arms and Flag sequence as templates. Then donor DNA was cloned into pBackZero-T vectors and pXRCC6-Donor vector was obtained.Then the two vectors were co-transfected into HeLa cells. Western blot was used to analyze the inserted efficiency. PCR and Western blot were used to analyze Flag tag which were inserted into C-terminus of XRCC6 gene in the genome and obtained XRCC6-Flag cell lines. These results provided a cell model for study of the function of XRCC6 gene.In this study, we succeed in editing Drosha/XRCC6/RNase L genes in genome and obtained stable HEK293 and HeLa cell lines respectively. Our study provides a foundation for further study of the function of these genes.