A CRISPR/Cas9-based Screening For Non-homologous End Joining Inhibitors And Study On Their Regulation In DNA Double Strand Break Repair

Purpose: Non-homologous end joining(NHEJ)is the major pathway responsible for the repair of ionizing radiation(IR)–induced DNA double strand breaks(DSB).The efficiency of NHEJ in tumor cells determines the sensitivity to IR-induced cytotoxicity.Therefore,targeting NHEJ pathway might hold the promise for improving the outcome of cancer radiotherapy.In this study,a cell-based screening for NHEJ inhibitors was presented by using the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system and high-resolution melting(HRM)analysis.Armed with the assay,an approved drug library was screened,and a collection of compounds were identified to be novel NHEJ inhibitors.Further experiments in vitro verified the suppressive effects on DSB repair and the radiosensitizing function of these drugs.And the mechanisms of their regulation in DSB repair were further clarified.Methods:(1)Firstly,A CRISPR/Cas9 plasmid containing the sg RNA sequence targeting HPRT or HBEGF gene was designed and constructed.After transfection of the plasmid into HEK293 T cells,the targeted site was amplified from genomic DNA by using PCR.The disruption of the HPRT gene or the NHEJ repair of the target site could be validated by Sanger sequencing data and T7E1 assay.After that the real-time PCR-amplified fragments containing the targeted site were delivered to HRM data analysis.To further examine the robustness of HRM analysis as a quantitative approach,a range of sg HPRT plasmid were transfected into HEK293 T cells after mixed with control plasmid.(2)Different cell lines were transfected with different CRISPR/Cas9 plasmids to obtain diverse values of △Fmax.The scheme which gained the highest △Fmax values would be chosen for the following screening.Cells were transfected with CRISPR/Cas9 plasmid for 5 hours,and then the cells were trypsinized and seeded in 96-well plates,compounds were added into the individual wells of the 96-well plates,and incubated for 24 hours.Then genomic DNA was extracted by a rapid Chelex 100-based method,and targeted locus was amplified and subjected to HRM analysis.Two DNA-PKcs inhibitors NU7441 and KU-0060648 were added in different concentration to confirm the feasibility of the screening workflow before the high-throughput screening.Finally,the compounds would be identified for effective NHEJ inhibitors,which could significantly decrease △Fmax values.(3)The cytotoxic effect of these compounds were evaluated by CCK-8 cell viability assay.The radiosensitizing effects of candidate compounds were measured via colony formation assay.To determine whether these compounds could block the DNA repair of the IR-induced DSBs,Neutral comet assay and cell cycle analysis were performed postirradiation,and immunofluoresence assay was carried out to quantify γ-H2 AX 、53BP1 foci in 12 h post-IR.Next,A well established HR/NHEJ assay(e GFP/I-SceⅠreport system)was employed to measure the effect of these compounds on HR/NHEJ efficiency.The mechanisms of their regulation in DSB repair were uncovered by using DNA-PK activity assay and western blot.Results:(1)HRM analysis was a quantitative means to determine the CRISPR/Cas9 plasmid-induced mutation.A length of 402 bp DNA sequence covering the targeted locus was amplified and subjected to T7E1 assay,which could digest the mismatched heteroduplex formed by the hybridization of the wild-type and mutant HPRT gene and generate two smaller fragments(approximate 130 bp and 270 bp).Disruption of the HPRT gene could also be validated by the overlapping peaks in the Sanger sequencing data.HRM analysis clearly revealed the disparity of the melting curve between sg HPRT treated cells and the control group.The melting curves were converted into a subtractive difference plot to show the fluorescence difference to the references(control plasmid–transfected cells).The maximum value of △F(the relative fluorescence difference)was used to determine the mutation frequency of the CRISPR/Cas9 plasmid induced NHEJ.Then a range of sg HPRT plasmid were mixed with control plasmid to provide different mutation frequency(0%、25%、50%、60%、70%、80%、90%、100%)and transfected into HEK293 T cells.HRM analyses showed the gradual shift of the subtractive fluorescence difference(△F)corresponding to the mutation efficiencies induced by different proportions of CRISPR/Cas9 plasmids.Importantly,the logarithm of △Fmax and the mutation efficiency displayed a strong positive linear correlation(R2=0.957).(2)Screening of an approved drug library including 1540 compounds.As an elevated △Fmax value might improve the sensitivity and resolution of the assay,the screening was performed using the sg HPRT plasmid and HEK293 T cells,by which the highest △Fmax value could be achieved.Both of the two DNA-PKcs inhibitors declined the△Fmax value in a concentration-dependent manner,implying the reduction of NHEJ-mediated mutations.A significant correlation between the concentrations and the logarithm of △Fmax values were observed(R2=0.987/0.961).Multiple candidate drugs from 1540 compounds were identified as effective NHEJ inhibitors.Concerning the potency of the NHEJ-inhibiting effects and their cytotoxicity,we next focused on ouabain and penfluridol in the following validating investigation.(3)Candidate drugs of NHEJ inhibitors impair IR-induced DSB repair to aggravate the cytotoxicity of IR.Ouabain and penfluridol were identified as effective NHEJ inhibitors,which could significantly decrease △Fmax values under the non-cytotoxic concentrations.Clonogenic assay revealed that the survival fractions of Hela and T98 G cells treated with ouabain and penfluridol were significantly lower than that of parental cells.The γH2AX and 53BP1 foci of ouabain or penfluridol-treated cells failed to resolve after 12 hours postirradiation compared with the control cells.Consistently,in the presence of ouabain or penfluridol,the DNA content in the comet tails was significantly increased.The propertion of cells at G2/M phase induced by IR was also elevated in the treatment of ouabain or penfluridol.(4)Candidate drugs of NHEJ inhibitors might interrupt DSB repair by preventing DNA-PKcs from being activated.NHEJ and HR reporter plasmid assay showed that both NHEJ and HR activities were suppressed by ouabain treatment,while only NHEJ activity seemed to be affected by penfluridol treatment.Western blotting analysis further revealed that ouabain or penfluridol treatment significantly restrained the expression of phosphorylated DNA-PKcs induced by IR.Moreover,they might not suppress the activation of ATM in response to IR,as well as its downstream target,Chk2.However,these NHEJ inhibitors did not seem to interact directly with DNA-PKcs,because they failed to show any inhibitory effects on DNA-PK kinase activity in vitro.Conclusion: As a major pathway to repair IR-induced DSBs in the mammalian cells,NHEJ could result in mutation,deletion or insertion of the nucleotides.Relying on the error-prone nature of the NHEJ-mediated ligation of the site-specific DSB induced by Cas9 nuclease,the present study demonstrated an innovative screening approach looked for NHEJ inhibitors by using the CRISPR/Cas9 system and HRM analysis.Next,an approved drug library containing 1,540 compounds was interrogated by using this screening strategy.The results identified ouabain,a cardiotonic agent,and penfluridol,an antipsychotic agent,have the capacity to restrain NHEJ activity.Further experiments in vitro revealed the radiosensitizing effects of these compounds and they might interrupt DSB repair by preventing DNA-PKcs from being activated indirectly.