Optimizing The Efficiency Of CRISPR System To Improve Gene Knockout Of Primate Embryos

CRISPR/Cas9 is a simple,highly efficient RNA-based genome editing technology widely used in plant and animal genome engineering.In recent years,Cas9 m RNA and sg RNA have been injected into cynomolgus fertilized egg cytoplasm to develop gene knockout animals.Non-human Primates are potential human diseases animal model.However,non-human primates are not preferred due to their long gestation period and the high rearing expanses.Additionally,it is difficult to obtain cynomolgus embryos;therefore it takes a long time to produce genetically modified embryos and fetuses.Under the premise of difficult to change the physiological characteristics of cynomolgus monkeys,the important way to reduce the cost and improve the efficiency of genetically modified monkeys is to improve the efficiency of CRISPR/Cas9 editing.Choosing efficient sg RNA to optimize the editing efficiency of CRISPR/Cas9 has become the main research goal of this thesis.We designed two consecutive experiments,first we predicted the basic trend of sg RNA sequences,on the basis of which two sg RNAs with high efficiency differences were tested on cynomolgus embryos,and secondly,C-terminal-binding protein interacting protein(Ct IP)was studied on Cas9 and has a low effect of active sg RNA efficiency.Experiment 1: First,we tested in vitro activity,and 8 active and one inactive sg RNA were selected.Six high efficient sg RNAs were injected into the embryos,and sg RNA(50ng/?L)and Cas9 m RNA(100 ng/?L)were mixed.Their efficiency was measured and compared with the efficiency scores on the common sg RNA design website.We found that sg RNA may have species differences.67 sg RNAs tested on cynomolgus embryos and statistically analyzed.A trend analysis of sg RNAs in primate embryos was obtained.Two sg RNAs tested on cynomolgus embryos and monoclonal results indicated that sg RNA 2activity was significantly higher than sg RNA 3 Off-target analysis of sg RNA 2 performed.The success of the experiment demonstrates that our trend analysis can screen for highly active sg RNAs and is not directly related to off-target.Experiment 2: For low-editing efficiency sg RNA analysis,it suspected that the DNA repair mechanism with the sister chromosome as a template,resulting in the failure of gene editing.This study designed to improve CRISPR/ Cas9 editing efficiency by adding Ct IP protein to improve the way of non-homologous end repair.For the first time,we mixed 300ng/?L of Ct IP protein into the vector and did not detect low activity sg RNA-T work.We mixed 500 ng/?L of Ct IP protein into the vector for the second time and still did not detect low activity sg RNA-T work.We collected 1 cell and 2 cells embryos into one tube and performed PCR sequencing,and no activity was detected.The experimental results demonstrate that Ct IP protein does not improve the editing efficiency of CRISPR after using low activity sg RNA.We obtained a trend analysis on the design of efficient primate embryo sg RNAs and explored the editing efficiency of Ct IP molecules in embryos with the CRISPR system.These results provide a direction for increasing the activity of the CRISPR system in primate embryos.We hope that with the support of more data,we will combine a more relevant report that has been reported so far to obtain a more complete activity scoring method for sg RNA in cynomolgus embryos.This greatly reduces uncertainty and extends the practical possibilities of cynomolgus monkey genome engineering.