Precise And Predictable Dna-fragment Editing By CRISPR/Cas9

Chromosomal rearrangements including large DNA-fragment inversions,deletions,and duplications by Cas9 with paired sg RNAs.Early studies used the programmable nucleases ZFN and TALEN to induce concurrent DSBs in the genome,generating chromosomal rearrangements of large DNAfragment,yet the low efficiency and time-consuming operation making them not widely used.The Type II CRISPR/Cas9 system derived from bacteria and archaea is an emerging genome editing technique and has been widely used in the chromosomal rearrangements of large DNA-fragment of eukaryotic cells.DSBs repaired by NHEJ pathway are stochastic and often produce indel mutation at the junction of the DNA-fragment editing,which also reduces the efficiency of precise DNA-fragment editing.Different programmable nuclease-mediated DNA cleavage could generate different configurations of DNA ends.As complex types of indel mutations are determined by repairing of DSB ends structure and DNA repair mechanism involved in chromosomal rearrangements,understanding the DNA cleavage mechanism of Cas9 nuclease and clarifying the DNA repair mechanism that induces chromosomal rearrangements are prerequisites for the precise and predictable CRISPR DNA-fragments editing.In this paper,we developed a CRISPR-induced DNA repair protein screening system to disrupt the DNA repair protein of alt-NHEJ repair pathway,and found that Ct IP and FANCD2 proteins can be used as targets to inhibit the activity of alt-NHEJ pathway,thereby promoting precise DNA-fragment deletion with different size of DNA-fragment,in the different loci,or with open or closed chromatin regions.Next,in order to further reveal that the c-NHEJ pathway is an accurate repair pathway,we inactivated XRCC4 and DNA Ligase IV repair protein involved in the c-NHEJ pathway,and found that the efficiency of precise deletion of large DNA-fragments was significantly reduced,indicating that the alt-NHEJ pathway competes with the c-NHEJ pathway for the repair of DNA DSBs.In addition,the resection activity of Ct IP protein is dependent on the phosphorylation of the active site.3AP,a small molecular inhibitor,could inhibit the phosphorylation of Ct IP protein.3AP treatment effectively improves the efficiency of precise ligation of large DNA-fragments deletion.Finally,we screened Ct IP mutant cell clones and found that Ct IP mutant cell clones could enhance precise ligation of large DNA-fragments deletion.3AP treatment on the Ct IP mutant cell clones further improves the precise DNA-fragment deletion.In summary,we can achieve precise DNAfragment deletion by disruption of Ct IP.Using the CRISPR-mediated chromosomal rearrangements and highthroughput sequencing technology,we could analyze the inserted nucleotides at the junctions of DNA-fragment editing of deletions,inversions,and duplications and characterize the cleaved products,and we found that nucleotide insertions are nonrandom and are derived from the sequences upstream of one PAM site or equal to the combined sequences upstream of both PAM sites with predicted frequencies.We speculated that the nonrandom nucleotide insertions resulted from the disequilibrium cleavage of the Cas9 nuclease.In-vitro cleavage assay revealed that the Ruv C nuclease cleaves the non-complementary strand with a flexible scissile profile upstream of the-3 position of the PAM site,generating double-strand break ends with 5? overhangs of 1-3 nucleotides.Furthermore,we screened engineered Cas9 nucleases with mutations of amino acid residues in the two linker regions.We found Cas9 variants could induce a flexible cleavage pattern.Thus,CRISPR DNA-fragment editing combined with high-throughput sequencing analyzing the cleavage pattern with different target sequences could facilitate precise and predictable DNAfragment inversions,deletions,and duplications.Finally,with this precise and predictable CRISPR DNA-fragment editing method,we inverted the Pcdh enhancer element,revealing that inversion of one single CBS at topological domain boundary can reshape long-distance chromatin-looping interactions between the distal enhancer and its target promoter.