Adopting Amplification Fragment Digestion Analysis To Detect Homology Recombination Efficiency Of Genome Editing

With the rapid development of genome editing technology,people can edit and modify the genomic sites at their will to achieve knocking out/knocking in target genes.Genome editing technology is a method to research gene function,produce transgenic animals and cure gene diseases.It also can modify gene to help us to understand the internal structure of genes,the relationships between gene function and biological phenotype by means of molecular biology.The CRISPR/Cas9 system is a new technology with the advantage of higher editing efficiency,simple processing and lower cost which has been using in human cells and model organisms,such as zebra fish,drosophila and mice.With the rapid development of CRISPR/Cas9 system,the therapy of human illnesses and cultivation of new plants/animals species can achieve significant changes.In somatic cell,the homologous recombination efficiency is too low to satisfy the demand of target gene precise repairing when genome double-strand break occurred.The level of the homologous recombination efficiency is closely related to the sites and the size of homologous recombination fragments.In previous studies,it was found that,with the increase of homologous arms,the homologous recombination efficiency is gradually increased.The study was designed on the basis of human 293 T cells,aiming to verify homologous fragment size on the effect of homologous recombination efficiency.It has been used CRISPR/Cas9 technology to induce genome double-strand break,repairing by different sizes of homologous recombination fragment(250 bp,500 bp,750 bp,1 kb,1.5 kb,2 kb).The result shows that with the increase of homologous fragment,homologous recombination efficiency are improved as well,but the increasing amplitude are not even.In some sites of detection,we also found that homologous recombination efficiency with the increase of homologous fragment size showed the first decrease after the increase trend.This research has established a method named AFDA(amplification fragment digestion analysis),which can use molecular biology technology to detect homologous recombination efficiency.Compared to the method in the eLIFE paper-with a 200 bp homologous fragment recombination repairing,AFDA can perform any length of homologous fragments detection.And our consequences show that the results of small fragments are not homogeneous.For different sites,the efficiency of short fragments is not as more reliable as the recombination efficiency of long fragments,and it cannot represent the real recombination efficiency of the site.On the detection of HR efficiency,we adopt three different methods,which are agarose gel electrophoresis,polyacrylamide gel electrophoresis and Agilent biological chip.Three methods all can be used,but for some small efficiency difference sites,agarose gel electrophoresis cannot have a good resolution.Polyacrylamide gel electrophoresis has a good resolution,but it needs to take a long time.By comparing,Agilent biological chip has significant advantages,which are high efficiency,high stability and high analysis rapidity.In addition,our laboratory verify small molecular on the effect of homologous recombination efficiency in cas9 protein stable expression cells,by adding different types of small molecular.The results show that the small molecular can improve cell?s homologous recombination efficiency.In general,the study will provide a method for detecting HR efficiency,so as to provide supports for screening high efficient sites,moreover,provide theoretical knowledge for transgenic animals production and gene therapy.