Using CRISPR/Cas9 System For Gene-Modified Pigs Generation And Cis-elements Annotation In Human Cells

In recent years, the widely popular CRISRP/Cas9 system, guided by RNA, which could specifically target and modify DNA, dramatically enhanced our ability in genome engineering. Since 2012, Jinek and others found the Cas9 protein in Type Ⅱ CRISPR/Cas system from Streptococcus pyogenes, could guide by RNA and digest DNA specifically in tubes. In the next few years, CRISPR/Cas9 has been widely used in DNA mutation, transcription regulation, epigenome modification, locus labeling of chromosome and high-throughput screening, which achieved many remarkable results.Pigs are considered as one of the most important economic species in agriculture, besides, due to their similarities with humans in anatomy and physiology, they are wonderful model animals in biomedical research and ideal donors for providing organs for xenotransplantation in clinical trials. Therefore, genome engineering in pigs has great benefits for economics, researches and clinical treatments. Here we used co-microinjection of Cas9 mRNA and sgRNA into one-cell stage zygotes of Chinese Bama miniature pigs, successfully achieved Npclll KO pigs. It is worth noting that the efficiency of Npclll mutation is 100%. Meanwhile, we analyzed Npc1l1 mutations in various somatic tissues and ovaries, indicating the mutations could be transmitted to offspring through germ cells. The off-target effect of CRISPR/Cas9 is an important bio-safety problem which cannot be overlooked. Especially off-target effect may cause dangers when applied in genome engineering for agriculture and clinical treatments. We screened potential off-target sites which are most similar to Npc1l1 sgRNA in pig genome by SeqMap tool and determined 8 candidates. However, we didn’t find any mutations in them. In addition to using CRISPR/Cas9 for generating gene-modified pigs, we also took advantage of TALEN to achieve B2m mutated pigs for comparison, as well as validated the heritance of the mutations. The cell surface of B2m knockout pigs lack MHC1, skin grafts from these donors to mice exhibited remarkably prolonged survival, which make pigs as further possible donors for xenotransplantation in the future.Besides using CRISPR/Cas9 system for gene knock-out, we also tried exploring the usefulness of dCas9 in genome engineering. Qi and others demonstrated that dCas9/sgRNA complex could bind to CDS region for blocking transcription elongation of RNAP through steric hindrance, which depress gene expression in prokaryotes. This enlightened us to take advantage of the high binding affinity of dCas9/sgRNA complex with target site, combine cis-elements by competing trans-factors, and abolish their function. To prove our hypothesis, human tet-on MAVS transgenic 293T cell was used. Upon DOX administration, MAVS was robustly induced, mimicking virus-trigged signaling, which would activate NF-kB IRF3/7 and cJun/ATF2 concurrently, these transcription factors could activate IFNB1 expression for starting antivirus function, which’s a classical cell signaling passway. We utilized dCas9/sgRNA specifically targeting the binding sites of NF-kB IRF3/7 or cJun/ATF2 in IFNB1 promoter, competed these factors and abolished their function, achieved the aim for blocking cell signaling passway. It’s the first time to prove CRISPR/Cas9 as a tool for functional annotation of endogenous cis-acting elements. This finding established new method for functional genomic study, as the same time displayed a new application of CRISPR/Cas9 in genome engineering.