Efficient In Vitro And In Vivo Deletion Of Large DNA Fragments By CRISPR/Cas9

Traditional homologous recombination strategy is widely applied to delete one or more exons in a gene locus to inactivate gene function. But this knockout strategy is limited in large fragment deletion. Recently new genome editing tools such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system were developed, which open the potential to manipulate large genomic fragment. To demonstrate the potential, here, we applied the CRISPR/Cas9 system to delete large fragment of noncoding genes. Firstly the Cas9 protein and sgRNAs were used to cut pEGFP-N1 plasmid in tubes. The results showed, the efficiency of one sgRNA-mediated cleavage and two sgRNAs-mediated fragment deletion were almost 100% supplied with enough Cas9 and sgRNA, demonstrating the possibility. We further optimized the reaction condition of this in vitro CRISPR/Cas9 system. With the success in vitro, we sought out to extend the strategy to in vivo application. A lentivirus-infected 293T cell line which stably expresses mCherry-P2A-EGFP is developed to report the cleavage efficiency of CRISPR/Cas9 system in human cells. The sgRNAs targeting at the CDS of mCherry were transfected together with Cas9 plasmids into report cells. As expected, the target fragment was deleted with high efficiency. With this tool, we further successfully generated a large fragment deletion in long noncoding RNA gene, Sox2ot, in mouse embryonic stem cell line. The 2.4 kb fragment deletion resulting in disrupted Sox2ot expression and disturbed ES cell proliferation and differentiation. Furthermore, by microinjection of Cas9 mRNA and sgRNA into mouse zygotes, a 23 kb fragment of lncRNA, Rian, was successfully deleted. The deletion efficiency was as high as 33.3% and the mutation could be transmitted to offspring as expected. Taken together, a strategy for large fragment deletion has been successfully established, which open the potentials in molecular cloning, developing genome-editing cell lines and animal models, and other related applications.