Non-homologous End Joining Frequency Research Articles

Comparison of CRISPR-Cas9-mediated megabase-scale genome deletion methods in mouse embryonic stem cells.

The genome contains large functional units ranging in size from hundreds of kilobases to megabases, such as gene clusters and topologically associating domains. To analyse these large functional units, the technique of deleting the entire functional unit is effective. However, deletion of such large regions is less efficient than conventional genome editing, especially in cultured cells, and a method that can ensure success is anticipated. Here, we compared methods to delete the 2.5-Mb Krüppel-associated box zinc finger protein (KRAB-ZFP) gene cluster in mouse embryonic stem cells using CRISPR-Cas9. Three methods were used: first, deletion by non-homologous end joining (NHEJ); second, homology-directed repair (HDR) using a single-stranded oligodeoxynucleotide (ssODN); and third, HDR employing targeting vectors with a selectable marker and 1-kb homology arms. NHEJ-mediated deletion was achieved in 9% of the transfected cells. Inversion was also detected at similar efficiency. The deletion frequency of NHEJ and HDR was found to be comparable when the ssODN was transfected. Deletion frequency was highest when targeting vectors were introduced, with deletions occurring in 31-63% of the drug-resistant clones. Biallelic deletion was observed when targeting vectors were used. This study will serve as a benchmark for the introduction of large deletions into the genome.

P.F508del editing in cells from cystic fibrosis patients.

Development of genome editing methods created new opportunities for the development of etiology-based therapies of hereditary diseases. Here, we demonstrate that CRISPR/Cas9 can correct p.F508del mutation in the CFTR gene in the CFTE29o- cells and induced pluripotent stem cells (iPSCs) derived from patients with cystic fibrosis (CF). We used several combinations of Cas9, sgRNA and ssODN and measured editing efficiency in the endogenous CFTR gene and in the co-transfected plasmid containing the CFTR locus with the p.F508del mutation. The non-homologous end joining (NHEJ) frequency in the CFTR gene in the CFTE29o- cells varied from 1.25% to 2.54% of alleles. The best homology-directed repair (HDR) frequency in the endogenous CFTR locus was 1.42% of alleles. In iPSCs, the NHEJ frequency in the CFTR gene varied from 5.5% to 12.13% of alleles. The best HDR efficacy was 2.38% of alleles. Our results show that p.F508del mutation editing using CRISPR/Cas9 in CF patient-derived iPSCs is a relatively rare event and subsequent cell selection and cultivation should be carried out.

Injection of an SV40 transcriptional terminator causes embryonic lethality: a possible zebrafish model for screening nonhomologous end-joining inhibitors.

IntroductionDNA repair by the nonhomologous end joining (NHEJ) pathway promotes tumor recurrence after chemotherapy and radiotherapy. Discovery of rapid and high-throughput techniques to screen for an effective NHEJ inhibitor drug is imperative for the suppression of NHEJ during tumor treatment. However, traditional screening methods are too cumbersome to meet the current need. Zebrafish is an ideal model for drug screening due to the specificity of its early embryonic development and similarity of tumor cell generation. By exploiting the high frequency of NHEJ in early embryonic development, we established a model that uses a transcriptional terminator signal fragment from the Simian virus 40 (SV40) to cause embryonic lethality. SV40 fragment-induced embryonic lethality was alleviated by 5,6-bis ((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol or C18H14N4OS (SCR7), an NHEJ inhibitor.Materials and methodsA 122 bp SV40 terminator fragment (10 ng/µL) was microinjected into zebrafish zygotes. SV40 fragment integration into the zebrafish embryonic genome was detected by Southern blot using a DNA probe for the SV40 terminator. Embryonic lethality rates were observed 24 and 48 h after microinjection. A nonhomologous recombinant inhibitor, SCR7 (5 µM), was used to alleviate embryonic lethality.ResultsMicroinjection of zebrafish embryos with the SV40 terminator fragment (10 ng/µL) caused a progressive increase in mortality over time. Using Southern blots, we confirmed that SV40 terminator sequences were integrated into the zebrafish embryonic genome. This phenomenon was effectively alleviated by addition of SCR7.ConclusionInjection of an SV40 terminator into zebrafish embryos may cause embryonic lethality due to NHEJ during early zebrafish development. The high mortality of zebrafish embryos could be alleviated by using the NHEJ inhibitor, SCR7. The zebrafish model presented here is simpler and more convenient than traditional methods of screening for NHEJ inhibitors and can be utilized in large-scale drug screens for NHEJ inhibitors and for the development of novel anticancer drugs.

Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing.

BackgroundThe ability to modify the genome of any cell at a precise location has drastically improved with the recent discovery and implementation of CRISPR/Cas9 editing technology. However, the capacity to introduce specific directed changes at given loci is hampered by the fact that the major cellular repair pathway that occurs following Cas9-mediated DNA cleavage is the erroneous non-homologous end joining (NHEJ) pathway. Homology-directed recombination (HDR) is far less efficient than NHEJ and makes screening of clones containing directed changes time-consuming and labor-intensive.MethodsWe investigated the possibility of pharmacologically inhibiting DNA-PKcs, a key player in NHEJ, using small molecule inhibitors (NU7441 and KU-0060648), to ameliorate the rates of HDR repair events. These compounds were tested in a sensitive reporter assay capable of simultaneously informing on NHEJ and HDR, as well as on an endogenous gene targeted by Cas9.ResultsWe find that NU7441 and KU-0060648 reduce the frequency of NHEJ while increasing the rate of HDR following Cas9-mediated DNA cleavage.ConclusionsOur results identify two small molecules compatible for use with Cas9-editing technology to improve the frequency of HDR.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0215-6) contains supplementary material, which is available to authorized users.

BRCA1 silencing is associated with failure of DNA repairing in retinal neurocytes.

Retinal post-mitotic neurocytes display genomic instability after damage induced by physiological or pathological factors. The involvement of BRCA1, an important factor in development and DNA repair in mature retinal neurocytes remains unclear. Thus, we investigated the developmental expression profile of BRCA1 in the retina and defined the role of BRCA1 in DNA repair in retinal neurocytes. Our data show the expression of BRCA1 is developmentally down-regulated in the retinas of mice after birth. Similarly, BRCA1 is down-regulated after differentiation induced by TSA in retinal precursor cells. An end-joining activity assay and DNA fragmentation analysis indicated that the DNA repair capacity is significantly reduced. Moreover, DNA damage in differentiated cells or cells in which BRCA1 is silenced by siRNA interference is more extensive than that in precursor cells subjected to ionizing radiation. To further investigate non-homologous end joining (NHEJ), the major repair pathway in non-divided neurons, we utilized an NHEJ substrate (pEPI-NHEJ) in which double strand breaks are generated by I-SceI. Our data showed that differentiation and the down-regulation of BRCA1 respectively result in a 2.39-fold and 1.68-fold reduction in the total NHEJ frequency compared with that in cells with normal BRCA1. Furthermore, the analysis of NHEJ repair junctions of the plasmid substrate indicated that BRCA1 is involved in the fidelity of NHEJ. In addition, as expected, the down-regulation of BRCA1 significantly inhibits the viability of retina precursor cells. Therefore, our data suggest that BRCA1 plays a critical role in retinal development and repairs DNA damage of mature retina neurocytes.

Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair

We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.

The ACF1 Complex Is Required for DNA Double-Strand Break Repair in Human Cells

DNA double-strand breaks (DSBs) are repaired vianonhomologous end-joining (NHEJ) or homologousrecombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in DSBs repair.

A recombination-based method to characterize human BRCA1 missense variants

Many missense variants in BRCA1 are of unclear clinical significance. Functional and genetic approaches have been proposed for elucidating the clinical significance of such variants. The purpose of this study was to evaluate BRCA1 missense variants for their effect on both homologous recombination (HR) and non homologous end joining (NHEJ). HR frequency evaluation: HeLaG1 cells, containing a stably integrated plasmid that allows us to measure HR events by gene conversion events, were transfected with the pcDNA3β expression vector containing the BRCA1-wild-type (BRCA1 wild type) or the BRCA1-unclassified variants (BRCA1-UCVs). The NHEJ was measured by a random plasmid integration assay. The assays suggested a BRCA1 involvement mainly in the NHEJ. As a matter of fact, the Y179C and the A1789T variant significantly altered the NHEJ activity as compared to the wild type, suggesting that they may be related to BRCA1-associated pathogenicity by affecting this function. The variants N550H and I1766S, and the mutation M1775R did not alter the NHEJ frequency. These data, besides proposing a method for the study of BRCA1 variants' effect on HR and NHEJ, highlighted the need for a range of functional assays to be performed to identify variants with altered function.

Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain ΔPcku70 shows up-regulation of genes from the HOG pathway

In Penicillium chrysogenum, the industrial producer of the beta-lactam antibiotic penicillin, generating gene replacements for functional analyses is very inefficient. Here, we constructed a recipient strain that allows efficient disruption of any target gene via homologous recombination. Following isolation of the Pcku70 (syn. hdfA) gene encoding a conserved eukaryotic DNA-binding protein involved in non-homologous end joining (NHEJ), a Pcku70 knockout strain was constructed using a novel nourseothricin-resistance cassette as selectable marker. In detailed physiological tests, strain DeltaPcku70 showed no significant reduction in vegetative growth due to increased sensitivity to different mutagenic substances. Importantly, deletion of the Pcku70 gene had no effect on penicillin biosynthesis. However, strain DeltaPcku70 exhibits higher sensitivity to osmotic stress than the parent strain. This correlated well with comparative data from microarray analyses: Genes related to the stress response are significantly up-regulated in the Pcku70 deletion mutant. To demonstrate the applicability of strain DeltaPcku70, three genes related to beta-lactam antibiotic biosynthesis were efficiently disrupted, indicating that this strain shows a low frequency of NHEJ, thus promoting efficient homologous recombination. Furthermore, we discuss strategies to reactivate Pcku70 in strains successfully used for gene disruptions.

Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks

Non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) is mediated by two protein complexes comprising Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/LigaseIV/XLF. Loss of Ku or XRCC4/LigaseIV function compromises the rejoining of radiation-induced DSBs and leads to defective V(D)J recombination. In this study, we sought to define how XRCC4 and Ku80 affect NHEJ of site-directed chromosomal DSBs in murine fibroblasts. We employed a recently developed reporter system based on the rejoining of I-SceI endonuclease-induced DSBs. We found that the frequency of NHEJ was reduced by more than 20-fold in XRCC4−/− compared to XRCC4+/+ cells, while a Ku80 knock-out reduced the rejoining efficiency by only 1.4-fold. In contrast, lack of either XRCC4 or Ku80 increased end degradation and shifted repair towards a mode that used longer terminal microhomologies for rejoining. However, both proteins proved to be essential for the repair of radiation-induced DSBs. The remarkably different phenotype of XRCC4- and Ku80-deficient cells with regard to the repair of enzyme-induced DSBs mirrors the embryonic lethality of XRCC4 knock-out mice as opposed to the viability of the Ku80 knock-out. Thus, I-SceI-induced breaks may resemble DSBs arising during normal DNA metabolism and mouse development. The removal of these breaks likely has different genetic requirements than the repair of radiation-induced DSBs.

Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting techniques. We show that cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways.

Alternative Pathways for the Repair of RAG-Induced DNA Breaks

RAG1 and RAG2 cleave DNA to generate blunt signal ends and hairpin coding ends at antigen receptor loci in lymphoid cells. During V(D)J recombination, repair of these RAG-generated double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway contributes substantially to the antigen receptor diversity necessary for immune system function, although recent evidence also supports the ability of RAG-generated breaks to undergo homology-directed repair (HDR). We have determined that RAG-generated chromosomal breaks can be repaired by pathways other than NHEJ in mouse embryonic stem (ES) cells, although repair by these pathways occurs at a significantly lower frequency than NHEJ. HDR frequency was estimated to be >or=40-fold lower than NHEJ frequency for both coding end and signal end reporters. Repair by single-strand annealing was estimated to occur at a comparable or lower frequency than HDR. As expected, V(D)J recombination was substantially impaired in cells deficient for the NHEJ components Ku70, XRCC4, and DNA-PKcs. Concomitant with decreased NHEJ, RAG-induced HDR was increased in each of the mutants, including cells lacking DNA-PKcs, which has been implicated in hairpin opening. HDR was increased to the largest extent in Ku70-/- cells, implicating the Ku70/80 DNA end-binding protein in regulating pathway choice. Thus, RAG-generated DSBs are typically repaired by the NHEJ pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways in these cells.

A genomics-based screen for yeast mutants with an altered recombination/end-joining repair ratio.

We recently described a yeast assay suitable for genetic screening in which simple religation nonhomologous end-joining (NHEJ) and single-strand annealing (SSA) compete for repair of an I-SceI-created double-strand break. Here, the required allele has been introduced into an array of 4781 MATa deletion mutants and each strain screened individually. Two mutants (rad52 and srs2) showed a clear increase in the NHEJ/SSA ratio due to preferential impairment of SSA, but no mutant increased the absolute frequency of NHEJ significantly above the wild-type level. Seven mutants showed a decreased NHEJ/SSA ratio due to frank loss of NHEJ, which corresponded to all known structural/catalytic NHEJ components (yku70, yku80, dnl4, lif1, rad50, mre11, and xrs2); no new mutants in this category were identified. A clearly separable and surprisingly large set of 16 other mutants showed partial defects in NHEJ. Further examination of these revealed that NEJ1 can entirely account for the mating-type regulation of NHEJ, but that this regulatory role was distinct from the postdiauxic/stationary-phase induction of NHEJ that was deficient in other mutants (especially doa1, fyv6, and mck1). These results are discussed in the context of the minimal set of required proteins and regulatory inputs for NHEJ.

Role of yeast SIR genes and mating type in directing DNA double-strand breaks to homologous and non-homologous repair paths

Eukaryotes have acquired many mechanisms to repair DNA double-strand breaks (DSBs) [1]. In the yeast Saccharomyces cerevisiae, this damage can be repaired either by homologous recombination, which depends on the Rad52 protein, or by non-homologous end-joining (NHEJ), which depends on the proteins yKu70 and yKu80 [2,3]. How do cells choose which repair pathway to use? Deletions of the SIR2, SIR3 and SIR4 genes – which are involved in transcriptional silencing at telomeres and HM mating-type loci (HMLα and HMRa) in yeast [4] – have been reported to reduce NHEJ as severely as deletions of genes encoding Ku proteins [5]. Here, we report that the effect of deleting SIR genes is largely attributable to derepression of silent mating-type genes, although Sir proteins do play a minor role in end-joining. When DSBs were made on chromosomes in haploid cells that retain their mating type, sir Δ mutants reduced the frequency of NHEJ by twofold or threefold, although plasmid end-joining was not affected. In diploid cells, sir mutants showed a twofold reduction in the frequency of NHEJ in two assays. Mating type also regulated the efficiency of DSB-induced homologous recombination. In MATa/MATα diploid cells, a DSB induced by HO endonuclease was repaired 98% of the time by gene conversion with the homologous chromosome, whereas in diploid cells with an α mating type (matΔ/MATα) repair succeeded only 82% of the time. Mating-type regulation of genes specific to haploid or diploid cells plays a key role in determining which pathways are used to repair DSBs.