Creation Of PDX-1 Gene Knockout Cynomolgus Monkeys Using CRISPR/Cas9 System

Diabetes mellitus(DM)is a heterogeneous group of metabolic disorders characterized by hyperglycemia and varying degrees of metabolic derangement as a result of b-cell insufficiency or impaired insulin secretion and/or action.It has become the third category of chronic non-communicable diseases that seriously endangers human health after cardiovascular and cerebrovascular diseases and tumor diseases.In recent years,the numbers of patients have been rising to 114 million in China,which is the highest in the world.Moreover,the numbers of diabetic patients are increasing in every country.By 2015,592 million people in the world are expected to have diabetic.The PDX-1 gene(pancreatic duodenal homeobox1)is a specific transcription factor that plays a key role in the development of the pancreas.It can affect the early development and late differentiation of the pancreas cells and lead to the occurrence and development of diabetes.With the development of gene editing technology,several genetically modified animal has been produced,such as mice,rat,rabbit,pig and sheep.However,none of them can completely imitate human diseases due to species difference.Non-human primates are highly similar(97%)with human therefore non-human primates has the potential to be used as potential human disease model.CRISPR/Cas9 technology has been wildly used in different research due to simple,convenient construction and high efficiency in gene editing.In this study,we constructed PDX-1 gene knockout monkeys using CRISPR/Cas9 technology,which provides a basis for the research of the molecular mechanism of diabetes.The specific experimental design,research content,and results are as follows.In this experiment,we designed eight sgRNAs on the PDX-1 gene sequence of the cynomolgus monkey on NCBI,and tested g RNAs activity in vitro.One to three sgRNAs and Cas9 mRNA were mixed and injected into the cytoplasm of cynomolgus embryos with ICSI method.The embryos were recovered in vitro after 5-7 days,and sgRNAs activity of the was examined on the embryos.A total of 27 embryos were recovered of which 21 embryos were developed to two more cells and subjected to TA monoclonal sequencing.Embryo transplantation performed using endoscopic minimally invasive surgery.After two weeks,the pregnant monkeys inspected with B-ultrasound.One female with abundant amniotic fluid and good physical condition was aborted around three month’s pregnancy,and amniotic fluid was extracted for identification.The monkeys pancreas tissue were dissected and fixed with formaldehyde and stored in-80℃.Genetic identify was introduced by scissoring monkey ears,extracting DNA of skin and TA monoclonal sequencing was proceeded.And then,pancreatic tissue was identified by HE staining.The activity of the designed target site was verified in vitro: the results showed that 6sgRNAs were active of which the sgRNA1,sgRNA2,sgRNA4,sgRNA5 on the first exon,sgRNA6 and sgRNA7 on the second exon.Embryo analysis showed that sgRNA1,sgRNA2,sgRNA4 and sgRNA7 have activity in embryo.A total of three female monkeys were pregnant of which one with twins and the other two with singletons.One of the pregnant female monkeys had a miscarriage in 117 d gestation(name 1010).The other one was naturally delivered baby in 168 d gestation(name 1126).The twins were born in 159 d by caesarean section.One of the twins survived for 4 hours(name 123101)and the other survived for 6 hours(name 123102).The genetic tests of the young monkeys were compared with those of the wild type monkey,the results showed that the 123101 and123102 monkey genes have homozygous mutations,and the 1010 monkey and the 1126 monkey have heterozygous mutations.From the anatomical point of view,the 1010 monkey and the 1126 monkey were found obvious pancreatic tissue,and the 123101 monkey and the 123102 monkey did not find obvious pancreatic tissue.Individually,the PDX-1 gene mutant monkey shape was smaller than that of the wild type monkey.There was no significant difference in pancreatic tissues between the 1010 monkey and WT monkey.It was difficult to find pancreatic islet tissues in the 1126 monkey pancreas compared with that of WT monkeys.Based on the above experimental results,we obtained two sgRNA with highly activity,and proved that the designed target site can be knocked out on the cynomolgus monkey PDX-1 gene.Moreover,the effect of a knockout the PDX-1first exon was better than that of the second exon,and espcas9 knockout efficiency is better compared to spcas9.the embryos co-injected with sgRNA1 and sgRNA2 were transplanted.Finally,two heterozygous knockout monkeys and two homozygous knockout monkeys were obtained.The corresponding technical system and established the PDX-1 gene knockout cynomolgus monkey were established to provide research ideas and reference for establishing diabetes animal model.The first step was to get a diabetic model of cynomolgus monkeys.