DNA Fragment Editing And Ligation Mechanism Of DNA Double-strand Breaks By CRISPR/Cas9

In recent years,genome editing technologies induced by ZFN,TALEN and Cas9 nucleases generating specific double strand breaks developed rapidly.Although genome editing technologies of ZFN and TALEN are very usefull,the complexity of their operating limits their wide application.However,CRISPR has the great advantages of easy design and convenient operation,and becomes the most competitive genome editing tool.Along with the completion of the human genome project and the human Encyclopedia of DNA elements,scientists found a lot of DNA regulatory elements and structural variations such as DNA fragment inversions and duplications in human genome.Futhermore,the genome of cancer cells exits frequently DNA fragment rearrangements.So,DNA fragment editing by CRISPR has an important role at the study of DNA regulatory elements,structural variations and disease research.In the second chapter,DNA fragment editing of DNA regulatory elements and gene clusters was generated by CRISPR with two sg RNAs in human cells and mice.Specifically,efficient DNA fragment inversions and deletions ranging in size from 35 bp to 807,480 bp could be generated in human cells and mice.In addition,DNA fragment duplications could also be achieved by CRISPR through trans-allelic recombination between double-strand breaks(DSBs)induced by Cas9 on two homologous chromosomes(chromatids).This method of DNA fragment editing is simple,less time-consuming,high efficient.Moreover,junctions of combinatorial inversions,duplications and deletions of the protocadherin gene clusters by Cas9 with four sg RNAs could be detected.DNA fragment editing with multiple sg RNAs has the high efficiency.Finally,the F1 mice with DNA fragment inversions and deletions successfully achieved from the founder F0 mice.We used the F1 mice and CRISPR cell lines to study the regulatory element of the Pcdha cluster and found a new role at the regulation of the Pcdhg cluster.To sum,the efficient DNA fragment editing by CRISPR should be useful in study of DNA regulatory elements and structural variations of mammalian genomes as well as vast numbers of gene clusters.In the third chapter,interrupting gene expression of Ct IP of NHEJ repair pathway could increase the efficiency of precise ligation of DNA fragment deletion.The Ct IP mutant cell lines by CRISPR also increase the efficiency of precise DNA fragment deletion.In addition,3-AP of small molecule drug inhibiting Ct IP function could improve the efficiency of precise DNA fragment deletion.Deletion junctions of interrupting gene expression of Ct IP responsible for cutting DNA double strand breaks have fewer mutations of bases deletions by Sanger sequencing and high-throughput sequencing,so manipulation Ct IP could achieve precise DNA fragment deletion and could be usefull for understanding the repair pathway.However,junctions of DNA fragment inversion have the special mechanism of ligation.One junction has the precise end ligations,the other one has the imprecise ligations with high proportional insertion mutations.Interrupting gene expression of Ct IP could not increase the efficiency of precise ligations of DNA fragment inversion.Precise DNA fragment inversion could achieved with specific combinations of PAM(NGG-NGG and CCN-CCN)by CRISPR.In a word,interrupting gene expression of Ct IP and using 3-AP could achieve precise DNA fragment deletion.Specific combinations of PAM(NGG-NGG and CCN-CCN)by CRISPR could achieve precise DNA fragment inversion.Futhermore,DNA fragment editing provides a usefull tool to study repair pathway in the cell.