Effect Of DNA Repair Proteins Mre11 And Sae2 On Genome Editing In Saccharomyces Cerevisiae

Genome editing technology is rapidly developed to be used in genome modification in recent years.The main principle is to induce DNA double strands break at the predesigned sites in the genome by using CRISPR(Clustered Regularly InterSpaced Short Palindromic Repeats)and Cas Protein Systems(CRISPR-associated systems),which are further repaired by non-homologous end joining(NHEJ)and homologous recombination(HR)of cells,thus resulting in random mutations and integrations exogenous fragments.When cells detect DNA breakage,DSB damage signal conduction leads to the response of DNA damage checkpoints,which can stimulate a series of life processes,such as cell cycle retention,autophagy,apoptosis,and so on,thus achieving efficient DSB repairs and ensuring smooth life processes.In this study,two key DSB repair proteins Mre11 and Sae2 were investigated.The effects of Mre11 and Sae2 gene knockout or mutation on the efficiency of both NHEJ-mediated random mutations and HR-mediated integration of exogenous fragments in Saccharomyces cerevisiae were determined.Thus,the mechanism of improving mutation efficiency by genome editing in Saccharomyces cerevisiae were discussed,and the mechanism of Mre11 and Sac2 affecting DSB repair were uncovered.First,the effects of Mre11 and Sae2 mutations on genome editing were evaluated at the cellular level.NHEJ-mediated genome editing was used,the growth curves of different mutant strains of Mre11 and Sae2 under genome edting conditions and non-edited conditions were measured to determine the inhibitory effects of different mutations on cell growth.At the same time,flow cytometry was used to analyze the impacts of genome editing on the cell cycle of wild type and mutant strains.No matter whether the intitial cells were synchronized or unsynchronized,DSB induced by both CRISPR/Cas9 and CRISPR/Cpf1 resulted in G2/M phase arrest.Furthermore,deletion or mutation of key DSB repair genes partially alleviated the cell cycle arrest.HR-mediated genome editing was targeted to the ADE2 site.The integration efficiency of exogenous fragments was found to be significantly reduced in MRE11 signle knockout and SAE2 and MRE11 double knockout strains.Second,the effects of Mre11 and Sae2 mutations on genome editing were evaluated at the transcriptional level.Transcriptome analyisis revealled that more stress response and redox pathway related genes were found in the 81 genes specifically up-regulated in CRISPR/Cas9 edited cells,while specific up-regulated genes in CRISPR/Cpf1 edited cells were enriched into DNA recombination.Using RT-PCR,the different gene expressions involved in DSB damage response were measured,the comparison of cell damage intensity between different types of genome editing was made clear,and the effects of different repair proteins on DSB damage response were analyzed.To sum up,this study investigated the function of two DSB repair proteins Mre11 and Sae2 in response to DNA damage and repair at both cellular and transcriptional levels.This study provided experimental basis for further analyzing DSB repair mechanism as well as improving editing effect and evaluating its safety.