Site-directed Modification Of Porcine Somatostatin Gene Using CRISPR/Cas9 Technology

CRISPR/Cas9 gene technology is a new editing techniques used in the study of gene function and transgenic engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a acquired immune defense system that the bacteria and archaea generated in a long evolutionary process. It is made of repeat sequences, spacer sequences (protospacers) and the cas protein family which is located in the upstream of repeat sequences. The system includes three kinds of types, the type ? CRISPR/Cas9 system structure is simple. In recent years, scientists have transformed it into a targeted genome editing tool in the mammalian genomic DNA. After transformation, this system only need a singel guide RNA(sgRNA) and a Cas9 protein. Cas9 identified the protospacer-adjacent motif sequences PAM(NGG) under the sgRNA to cause the DNA cleavage. Biological organism repaired the breaks that occurs insertions, deletions, and large fragments of DNA bases deleted, through its own inaccurate repair system. It achieved the orientation modify of a particular gene.Somatostatin (SST) secreted by the hypothalamus, is a kind of neuromodulation peptide that inhibits the secretion of growth hormone (GH). In the growth process, growth hormone (GH) is the core of regulating the growth of animals, its secretion is regulated by two hormones of hypothalamus. Growth hormone (GHR) releasing factor promotes GH secretion, SST suppresses its release. Besides inhibiting GH secretion, SST also can restrain thyroid stimulating hormone, insulin, glucagon, and gastric secrete element etc, it affects the function of the digestive system, inhibits the digestive tract to absorb nutrients such as glucose, thus inhibiting the growth of animals. The pig is closely connected with human life, pork is the main source of meat supply in China. Meanwhile, the pig is more closer to people in anatomy, tissue, physiology and nutrition metabolism, so it is also the ideal animal model of human disease. Thereby, the use of site-directed gene-modified pigs produced SST gene deletion can not only help improve the breeding in livestock production, improve pig growth rates, but also can be used as an experimental animal model to study the growth inhibition and other related diseases. In this study, five 20bp sgRNAs targeting exonl and exon2 of SST were designed, they were SST-sgRNA-g1, SST-sgRNA-g2, SST-sgRNA-g3, SST-sgRNA-g4 and SST-sgRNA-g5. Chemically synthesized, and then inserted them into linearized plasmid which expressed the sgRNAs and Cas9 protein together. Detecting the activities of the targets using the CRISPR/Cas9 technology, selecting the SST-sgRNA plasmids which had highly activity to achieve gene modification in two ways. One way:the plasmids which expressed the highly activity sgRNAs were extracted using the endo-free plasmid kit, and transfected into the PK15 cell through electroporation technology, then the stable transfection cell lines were selected by limiting dilution method, finally, the genomic DNA of cell was extracted and the target efficiency was detected by T7NE1 enzyme and Sanger sequencing. The other way:two complementary oligo DNA of sgRNA were synthesized and then annealed to a double-strand DNA (naturally dropped to room temperature after 5 minutes of 95?), then ligated to the expression vector pDR274 which was digested by Bsa I to form sgRNA expressing plasmid. DNA sequencing screened the correct pDR274-sgRNA plasmids which were the template of sgRNA in vitro transcription. Meanwhile, the Cas9 expression plasmid was linearized by Pmel and purified using phenol-chloroform extraction, then the linearized plasmid was dissolved into nuclease free water as a template for Cas mRNA transcription in vitro. Two sgRNA with Cas9 mRNA were mixed in a certain concentrition, then microinjected into oocytes of the pigs with a best RNA concentrition. After parthen ogenetic activation, the oocytes were cultured for 48h at 39?,5% CO2 in air, then the genomic DNA was obtained by the NP-40 lysis buffer, large fragmen ts deletions w ere preliminary screened by nested PCR amplification, then the large fragment deletions and single site base mutations were further detected by sequence analysis. The results as follows:1. The target nucleotide sequences were successfully inserted into the expected sites of vector and sequences were correct. The vector was successfully transcribed into sgRNA in vitro. Target DNA fragments were digested by Cas9/sgRNA in vitro, then compared with standard sgRNA 1 and sgRNA2 enzyme activity, the target of SST-sgRNA-gl, SST-sgRNA-g4 and SST-sgRNA-g5 had highly activities and the target SST-sgRNA-g4 had the highest activity. This could provide basis for the site-directed modification of SST gene in cell and embryo.2. The plasmid which expressed the sgRNA and Cas9 protein together, transfected into the PK15 cell using electroporation technology, the transfection efficiency was about 80%. The fluorescent clone cells of three groups were selected successfully by limiting dilution method, the group of SST-sgRNA-gl had 15 monoclonal cell lines, SST-sgRNA-g4 was 17 monoclonal cell lines and SST-sgRNA-g5 was 13 monoclonal cell lines. Each group was successfully extracted genomic DNA and PCR ampilification obtained the DNA sequence that included the target sites, the PCR products were digested by T7NE1 and sequence analysis. But the bases mutations were not detected.3. The plasmid of pDR274-sgRNA and the Cas9 expression plasmid pMLM3613 were constructed successfully. Obtaining the highly concentraction of sgRNA and Cas9 mRNA in vitro transcription. Adjusting Cas9 mRNA/sgRNA concentration ratio to select the best RNA injection concentration, finally, it was shown that Cas9 mRNA/ sgRNA concentration in 100 ng/uL-200 ng/uL range can obtain a higher rate of parthenogenetic embryo cleavage.4. Embryos were lysised by NP-40 buffer and extracted genomic DNA successfuLly. There were 25 parthenogenetic embryos were deleted the chromosomal DNA fragments in 100 embryos, the deletion efficiency was about 25%. In order to determine the large fragment deletions accurately, selecting the PCR products about 336 bp, Ta cloning and sequencing, at the same time, the PCR products of the other 75 embryos which were not deleted large fragments were also sequenced to detect the base mutation, it was found that 10 embryos occurded base mutations in the target sites, the mutation rate was about 7.5%.To sum up, by constructing SST genetically modified vectors, transfecting vectors into cells with electricity, microinjecting pigs mature oocyte, parthenogenetic activation and testing early embryo of total DNA, a new system of CRISPR/Cas9 knockout the SST gene of pigs was established on the embryo level. It was not noly provided a theoretical basis for the preparation of missing SST transgenic pigs, but also provided a new way for China's pig breeding and improvement.