Research On The Establishment Of Site Directed Knock-in Transgenic Cell Of Sheep Mesenchymal Stem Cells By CRISPR/Cas9 System

The CRISPR/Cas9 system has recently emerged as a powerful tool for preparation of transgenic animals and functional genomic study in mammals.Studies have shown that ROSA26 gene can encode a non-essential nuclear RNA in almost all organizations and become an important site of exogenous gene insertion.In order to establish the method of exogenous gene site-specific integration in ROSA26 targeting loci with homologous recombination mediated by CRISPR/Cas9 system,we designed a sheep ROSA26 locus sgRNA and its left and right homologous arms expression vector.We inserted the fluorescence reporter gene GFP in the ROSA26 locus of ovine umbilical cord mesenchymal stem cells by the CRISPR/Cas9,and then detected the homologous recombination of GFP gene by molecular biology method.The results obtained are as follows.1.The construction of sheep ROSA26 sgRNA/Cas9 expression vector.According to the gene sequence of sheep ROSA26 of Wang'spublication,we searched for acailable sgRNA sequence on Zhang Feng's website(http://crispr.mit.edu/),and then selected 1620-1778 bp out of the suequences for the study according to the score and the location.We combined the sgRNA with the PX330 vector by the method of point mutation and analyze the sequence,the ROSA26 sgRNA/Cas9 expression vector(sROSA26-sgRNA-1)was successfully constructed.2.Verification of sheep ROSA26 sgRNA/Cas9 expression vector.After transfection sROSA26-sgRNA-1 in the sUMSC,we detected the efficiency induced by Cas9 with T7E1.We analyze the agarose gel electrophoresis results according to the formula: the mutation rate = mutant band /(wild type + mutant type).As a result,the sgRNA worked at the frequency approximately 20%.The results showed that we successfully constructed the ROSA26 sgRNA/Cas9 expression vector.3.The constrction of sheep ROSA26 left and right homologous arm expression vector.According to the designed sgRNA sequence,we chose the primers of the left and right homologous arms in the upstream and downstream of the ROSA26 gene targeting site of sheep,and the length were 1093 bp and 928 bp.And then we combined the PCR product with DCDON-SH02 ROSA26.After the identification of PCR,enzyme digestion and sequence analysis,we we successfully constructed the homologous arm expression vector sROSA26-HA.4.The selection of positive clones and detection of homologous recombination.sROSA26-HA and sROSA26-sgRNA-1 were cotransfected into sUMSC by Lipofectamine2000,which were screend by puro 14 d,and obtained positive clones expressing green fluorescent protein.PCR was used to detect the insertion of exogenous gene in the cell clones.As a result,we targeted and resulted in homologous recombination in sUMSC ROSA26 locus successfully induced by CRISPR/Cas9.The results showed that we targeted the sheep ROSA26 locus successfully by CRISPR/Cas9 system and inserted GFP gene in the target site by homologous recombination.By this way,we established the method of exogenous GFP gene knock-in sheep umbilical cord mesenchymal stem cells mediated by CRISPR/Cas9.The results of this study provided the scientific basis for further study on the functional gene analysis of sheep and the preparation of transgenic sheep.