Gene Editing Of NLGN4X In Guinea Pig Cells Using CRISPR/Cas9 Technology

In recent years,the rapid rise of ZFN,TALEN technology,especially the CRISPR/Cas9 system,since the high efficiency,convenience,short cycle and low cost,it has been widely used to edit animal genomic,it also makes the study of the gene knockout model become more convenient.Although gene editing technology is widely used in mice,rat and many other mammalian species,it has not received much attention in guinea pigs.As a common experimental animal,guinea pigs is widely used in the studies on immunology and pharmacology.it is sensitive to hearing and smell,and is very sensitive to external stimuli.Therefore,it has great reference value for the diagnosis and treatment of mental diseases.In this study,the Neuroligin-4,X-linked gene(NLGN4X)of guinea pigs cell have been edited by CRISPR/Cas9 system efficiency.In this study,we isolate the guinea pig fetal tissue and obtain the guinea pig fibroblasts.the composition and transfection conditions of the cells have been optimized.The optimized primary cells maintained normal proliferation ability,it could meet the needs of cell cryopreservation and passage.At the same time,we select the first exon and the second exon of the NLGN4X gene in guinea pig genomic.By searching the target sequence,four pair of gRNAs(cN1,cN2,ctN1,ctN2)were designed to be constructed into the gene knockout carrier(RGNs).After being stained with puromycin,the genomic DNA of guinea pig fetal fibroblasts was detected by T7E1 enzyme digestion.The results showed that the editing efficiency of standard gRNA(cN1,cN2)on the target loci of NLGN4X gene was 29.5%and 53.8%respectively,and the editing efficiency of truncated gRNA(ctNl,ctN2)was 42%and 60.7%respectively(P<0.05).The above results showed that the CRISPR/Cas9 system was active in the gene editing of the guinea pig cells,indicating that the gene knockout carrier containing truncated gRNA could improve the efficiency of gene editing of the guinea pig cells.CRISPR/Cas9 technology can edit genome quickly and efficiently.it is suitable for gene knockout in guinea pig cells.The relevant data in this study provide efficient gRNAs for the construction of the guinea pig gene knockout model,which can be used to establish an animal model of NLGN4X knockout in guinea pigs.This study will help the pathological study,clinical treatment and drug development of ASD disease.