The B-cell Antibody Class Transfer Model Was Used To Study The A-EJ Repair Pathway

Maintenance of genomic integrity and stability is of prime importance for the survival of an organism.DNA double strand break(DSB)is considered as one of the most deleterious type of DNA damages,which can be generated by exogenous stresses,such as ionizing radiation(IR)or chemical agents,and endogenous stresses,such as endogenous oxidation or replication stresses.DSB repair pathways in mammals can be broadly classified into two categories,namely,homologous recombination(HR),which is dependent on homologous sequences,and non-homologous end joining(NHEJ).More recent experiments identified a new NHEJ pathway,termed alternative end-joining(A-EJ),which has been shown to play an important role in joining of DSBs in the absence of classical NHEJ(C-NHEJ)proteins.Especially,A-EJ can mediate up to 50%of wild-type(WT)IgH class switching recombination(CSR)levels in C-NHEJ deficient B cells,such as DNA Ligase Ⅳ(Lig4)deficient cell.Most alternative end-joining events reported so far appear to involve micro-homologous(MH)sequences.A-EJ activity mediates the majority of chromosomal translocations.Several putative A-EJ factors have been proposed,although results are mostly controversial.Especially the role of DNA ligase in the A-EJ process is still unclear.By CRISPR/Cas9 system,we generated mouse CH12F3 cell lines in which,in addition to Lig4,either Ligase Ⅰ(Lig1)or nuclear Ligase Ⅲ(Lig3)was completely ablated,which also representing the cells containing a single DNA ligase(Lig3 or Lig1,respectively)in their nucleus.Surprisingly,we found that both Lig1-and Lig3-containing complexes could efficiently catalyze A-EJ for CSR in IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes.However,only deletion of nuclear Lig3,but not Lig1,could significantly reduce the inter-chromosomal translocations in Lig4-/-cells,suggesting the unique role of Lig3,different from Lig1,in catalyzing chromosome translocation.Additional sequence analysis of chromosome translocation junction micro-homology revealed the specif-icity of different ligase-containing complexes.The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ.In addition,we used CSR as the model to do whole-genome scale CRISPR/Cas9 screening for A-EJ pathway factors in WT and Lig4-/-cells.Firstly,we generated WT and Lig4-/-mouse B cell line CH12F3 expressing Cas9 stably,and optimized the whole screening processes.High-throughput sgRNA screening was performed in these Cas9 expressing cells using optimized experimental conditions.Analysis of each sgRNA abundance in different cell populations was performed by MAGeCK assay,to choose the significantly reduced genes in the IgA positive population and increased genes in IgA/IgM double negative population.In the WT cells,the core C-NHEJ factors,such as DNA-PKcs and Lig4,and some known genes involved in the cytokine-mediated CSR process were screened out.Detection of the other candidate genes with unknown functions revealed that Brd9 and Fbxo28 deficiency can seriously affect cytokine-induced CSR.This result is currently being further validated.At the same time,high-throughput screening based on the Cas9/sgRNA-induced CSR model is also underway.