Study Of Molecular Design And Delivery Method On CRISPR/Cas9 Ribonucleoproteins With High-efficiency Homology-directed Repair Activity

The 2020 Nobel Prize in Chemistry was awarded to French scientist Emmanuel Charpentier and American scientist Jennifer Doudna in recognition of the discovery and development of a genome editing technology termed CRISPR/Cas9.It allows people to edit genes efficiently and to study gene functions more intuitively,showing a revolutionary impetus in various parts of life science research.However,accuracy and safety are the two major problems that currently limit its applications in vivo.To develop new methods and technologies for precise genome editing,we herein chose the Cas9 ribonucleoprotein(RNP)that has a controllable transfection dose and a short half life to perform researches.Firstly,we developed a nanoparticle material with low cost and high biocompatibility,which can can efficiently deliver Cas9 RNP and DNA repair donor together into cells,achieving homology-directed repair(HDR)-mediated precision repair.Using a "biological self-assembly" method established in the laboratory,we prepared the Etag-Cas9 RNP containing glutamate polypeptide(Etag)at the N-terminal.This RNP is rich in negative charges and can self-assemble into nanoparticles about 200 nm in diameter,through electrostatic interaction with the red fluorescent protein encapsulated by chitosan and the single-stranded DNA(ssDNA)repair template.This method achieved 77.6% delivery efficiency and 12.5% ??HDR-mediated genome editing efficiency,and produced effective genome editing in a variety of cell lines.Compared to the traditional liposome delivery method,this method not only shows considerable delivery efficiency and editing efficiency,but also has a simpler construction workflow and a better biocompatibility,demonstrating a wide application prospect.In nature,it has been shown that the most double strand breaks(DSB)produced by gene editing are recovered through non-homologous end repair(NHEJ).However,this pathway often causes deletions or insertions in the genome,resulting in uncontrollable repair.In contrast,the efficiency of homology-directed repair(HDR)pathway that can generate precise repairs is often low.In this study,the molecular design and optimization of Cas9 RNP were carried out to achieve a high HDR efficiency.The sh RNA sequence of Ligase 4 gene,a key factor in the NHEJ pathway,was introduced to the 3'end of g RNA in Cas9 RNP.We combined the Cas9 gene editing technology with RNAi interference technology,constructing the Cas-RNAi RNP which has an improved HDR efficiency.Currently,the experiments to verify the effect of HDR in genomes are still being repeated and optimized.In short,the methods demonstrated here can provide theoretical guidance and technical basis for the large-scale application of Cas9 RNP in gene therapy.