Research On CRISPR/Cas9 System Mediated Mouse RBM10 Gene Knockout

CRISPR/Cas9 system is the newest gene designated editing tool,which is developed from bacteria's resistance mechanism to the invasion of foreign DNA.Due to it's multiplex advantages such as simple construction,wide application range,high efficiency,low cost and so on,it is loved by lots of scientists and has been used successfully in many fields such as medicine and animal/plant breeding.The RBM10(RNA binding motif protein 10)gene,which is located on the X chromosome,is a member of the RBM gene family.The translation product participates in the regulation of apoptosis.Several studies have pointed out that the mutations of RBM10 gene may be related to certain diseases or cancers.In this study,a pair of Cas9 target sites that could efficiently delete RBM10 gene was screened in mouse fertilized eggs by single cell PCR technology,which provided a reference for the rapid acquisition of animal models of disease mutation or knockout.Single cell PCR is a technique that can be amplified simply by using the genetic material contained in a single cell as a template.How to obtain the genetic material in a single cell and prepare it as a high-quality template is a the key to the success of the whole reaction In this study,three different protease K cell lysates(SDS cell lysate,NP-40 cell lysate,and Tween-20 cell lysate)were first used to lyse mature single pig oocytes in vitro and the lysate was directly used as a template for amplification by Nested PCR.The final results showed that the NP-40 cell lysate had the best template quality with an amplification efficiency of 91%;followed by Tween-20 cell lysate with an amplification efficiency of 61.0%;No positive bands were detected in the SDS cell lysate product.By searching the NCBI gene library,the mouse RBM10 gene-related sequences were obtained,and the Cas9 target sites were predicted by Zifit software.The expression plasmid vectors of the corresponding target sites gRNA were constructed;The vector plasmid was used as a template of PCR reaction to get the template for sgRNA transcription in vitro.Using T7 vitro transcription kit to obtain the corresponding target site gRNA.The Cas9 mRNA and the purified target site gRNA were mixed and diluted,and finally the concentration of Cas9 mRNA was adjusted to200 ng/?L,the concentration of the target site gRNA was 30 ng/?L,The mixture was microinjected into the cytoplasm of mouse fertilized eggs.The detection efficiency of early embryos was determined by the single-cell PCR assay,which has been constructed in the previous period.The results showed that the amplification resultswere good and there were a large number of suspected positive-amplified fragments(100-200bp).Sequencing analysis was performed on the suspected positive fragments.The results showed that the long DNA fragment of about 2100 bp between the T1 and T2 target sites of the mouse RBM10 gene was successfully deleted.After statistical analysis,the deletion rate was approximately 81.9%.And there is no off-target phenomenon.