Microhomology-mediated End Joining Pathways Research Articles

Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks

BackgroundCRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks.ResultsUsing long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells.ConclusionsOur findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

DPPA5A suppresses the mutagenic TLS and MMEJ pathways by modulating the cryptic splicing of Rev1 and Polq in mouse embryonic stem cells

Genetic alterations are often acquired during prolonged propagation of pluripotent stem cells (PSCs). This ruins the stem cell quality and hampers their full applications. Understanding how PSCs maintain genomic integrity would provide the clues to overcome the hurdle. It has been known that embryonic stem cells (ESCs) utilize high-fidelity pathways to ensure genomic stability, but the underlying mechanisms remain largely elusive. Here, we show that many DNA damage response and repair genes display differential alternative splicing in mouse ESCs compared to differentiated cells. Particularly, Rev1 and Polq, two key genes for mutagenic translesion DNA synthesis (TLS) and microhomology-mediated end joining (MMEJ) repair pathways, respectively, display a significantly higher rate of cryptic exon (CE) inclusion in ESCs. The frequent CE inclusion disrupts the normal protein expressions of REV1 and POLθ, thereby suppressing the mutagenic TLS and MMEJ. Further, we identify an ESC-specific RNA binding protein DPPA5A which stimulates the CE inclusion in Rev1 and Polq. Depletion of DPPA5A in mouse ESCs decreased the CE inclusion of Rev1 and Polq, induced the protein expression, and stimulated the TLS and MMEJ activity. Enforced expression of DPPA5A in NIH3T3 cells displayed reverse effects. Mechanistically, we found that DPPA5A directly regulated CE splicing of Rev1. DPPA5A associates with U2 small nuclear ribonucleoprotein of the spliceosome and binds to the GA-rich motif in the CE of Rev1 to promote CE inclusion. Thus, our study uncovers a mechanism to suppress mutagenic TLS and MMEJ pathways in ESCs.

POLQ inhibition elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma via cGAS/STING signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination-repair (HR-repair) proteins in 20%-25% of cases. Defects in HR impart a specific vulnerability to poly ADP ribose polymerase inhibitors and platinum-containing chemotherapy in tumor cells. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we found that POLQ knockdown is synthetically lethal in combination with mutations in HR genes such as BRCA1 and BRCA2 and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates signaling of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING), leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to blocking tumor growth while concurrently activating the cGAS-STING signaling pathway to enhance tumor immune infiltration, highlighting what we believe to be a new role for POLQ in the tumor immune environment.

Abstract 512: Identification of SS008871, a novel Polθ inhibitor that effectively inhibits tumors with homologous recombination deficiency in vitro and in vivo

Abstract The repair of DNA double strand break (DSB) is crucial for genome stability and cell survival. There are three main DSB repair pathways: homologous recombination (HR), non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ). HR is a high-fidelity, error-free DSB repair pathway, and the dysfunction of HR confers cell genome instability and leads to tumorigenesis. HR deficiency is especially prevalent in gynecologic tumors, sharing about 10% population of all the gynecologic tumor patients. In situation of HR deficiency, MMEJ, in which DNA polymerase theta (Polθ) plays an essential role, is up-regulated to serve as a backup pathway for DSB repair. Several studies have proved that the inhibition of Polθ causes synthetic lethality with HR deficiency. Hence, Polθ emerges as a potential DNA damage repair target for the treatment of HR deficient tumors. Here we report a novel small molecular Polθ inhibitor, SS008871, which inhibits Polθ activity with an IC50 of 22 nM, and strongly inhibits cellular MMEJ pathway with an IC50 of single-digital nanomolar level. SS008871 strongly inhibits proliferation of HR deficient BRCA2-/- DLD-1 cells, and shows a >125× selectivity folds over DLD-1 parent cells as well as non-malignant cells. Besides, SS008871 elicits synergetic anti-proliferation activities in combination of a PARP inhibitor, olaparib on BRCA2-/- DLD-1 and MDA-MB-436 cells. In the BRCA2-/- DLD-1 xenograft model, SS008871 shows tumor growth inhibition as a single agent, and the combination of SS008871 and olaparib further results in tumor regression. Accordingly, the level of γH2AX, a common DSB marker, correlates well to the anti-tumor efficacy. In a human hematopoietic stem cells based in vitro hematotoxicity assay, SS008871 shows no significant inhibition on lineage-specific (myeloid, erythroid and megakaryocytic) cell differentiation and survival, suggesting the low hematotoxicity risk. In comparison, olaparib significantly attenuated hematopoietic stem cells on both differentiation and survival in the parallel assay, which is consistent with the hematological toxicity observed in human. Furthermore, there is no clinical abnormalities observed after a high dose treatment of SS008871 in mice, demonstrating that SS008871 is well-tolerated. Taken together, SS008871 is proved to be an encouraging Polθ inhibitor with good safety. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Feng Tang, Zhen Li, Wenqing Yang, Liting Xue, L Chen, Renhong Tang. Identification of SS008871, a novel Polθ inhibitor that effectively inhibits tumors with homologous recombination deficiency in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 512.

DNA double-strand break repair machinery in Penaeid crustaceans: A focus on the Non-Homologous End-Joining pathway.

DNA double-strand breaks (DSBs) are repaired through three major pathways: Non-Homologous End-Joining (NHEJ), Microhomology-Mediated End-Joining (MMEJ), and Homology-Directed Repair (HDR), each requiring a specific set of diverse proteins. Such pathways and their proteins have been studied in model organisms, including arthropods; however, DSB repair pathways are scarcely described in Crustacea, a taxon that includes the commercially valuable penaeid shrimps (Crustacea: Decapoda: Penaeidae). In this work, transcriptome and proteome databases of Penaeus vannamei and other Crustacea species were scrutinized for each protein of the NHEJ pathway. The structural and functional attributes of such proteins in penaeids were determined using bioinformatics. Additionally, the expression of the NHEJ-related Ku70, Ku80, DNA-PKcs, DNA ligase 4 (Lig4), and HDR- and MMEJ-related protein transcripts were assessed in P. vannamei gills, midgut gland, hemocytes, and muscle by RT-PCR. DSB repair protein transcripts were found expressed in the four assayed tissues, particularly in the gills and midgut gland. Among DSB repair proteins, all the analyzed transcripts of proteins related to the NHEJ pathway were present in gills. To the best of our knowledge, this is the first report on the expression of DSB repair proteins in Decapoda. Together, proteomic, transcriptomic, and expression data suggest the functionality of NHEJ, HDR, and MMEJ pathways in P. vannamei and other decapods. The information presented here contributes to understanding the response to DSB breaks in shrimps, describing possible outcomes in oxidative stress studies and also in the designing of gene editing strategies, which have not been developed in Penaeidae.

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51

DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2–4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

Endogenous CRISPR-assisted microhomology-mediated end joining enables rapid genome editing in Zymomonas mobilis

BackgroundZymomonas mobilis is a natural ethanologen with many desirable characteristics, making it an ideal platform for future biorefineries. Recently, an endogenous CRISPR-based genome editing tool has been developed for this species. However, a simple and high-efficient genome editing method is still required.ResultsWe developed a novel gene deletion tool based on the endogenous subtype I–F CRISPR-Cas system and the microhomology-mediated end joining (MMEJ) pathway. This tool only requires a self-interference plasmid carrying the mini-CRISPR (Repeat–Spacer–Repeat) expression cassette, where the spacer matches the target DNA. Transformation of the self-interference plasmid leads to target DNA damage and subsequently triggers the endogenous MMEJ pathway to repair the damaged DNA, leaving deletions normally smaller than 500 bp. Importantly, the MMEJ repair efficiency was increased by introducing mutations at the second repeat of the mini-CRISPR cassette expressing the guide RNA. Several genes have been successfully deleted via this method, and the phenotype of a σ28 deletion mutant generated in this study was characterized. Moreover, large fragment deletions were obtained by transformation of the self-interference plasmids expressing two guide RNAs in tandem.ConclusionsHere, we report the establishment of an efficient gene deletion tool based on the endogenous subtype I–F CRISPR-Cas system and the MMEJ pathway in Zymomonas mobilis. We achieved single gene deletion and large-fragment knockout using this tool. In addition, we further promoted the editing efficiency by modifying the guide RNA expression cassette and selecting lower GC% target sites. Our study has provided an effective method for genetic manipulation in Z. mobilis.

Poly ADP Ribose Polymerase Inhibitor Olaparib Targeting Microhomology End Joining in Retinoblastoma Protein Defective Cancer: Analysis of the Retinoblastoma Cell-Killing Effects by Olaparib after Inducing Double-Strand Breaks.

Retinoblastoma is the most common intraocular cancer in childhood. Loss of function in both copies of the RB1 gene is the causal mutation of retinoblastoma. Current treatment for retinoblastoma includes the use of chemotherapeutic agents, such as the DNA damaging agent etoposide, which is a topoisomerase II poison that mainly generates DNA double-strand breaks (DSBs) and genome instability. Unfaithful repairing of DSBs could lead to secondary cancers and serious side effects. Previously, we found that RB knocked-down mammalian cells depend on a highly mutagenic pathway, the micro-homology mediated end joining (MMEJ) pathway, to repair DSBs. Poly ADP ribose polymerase 1 (PARP1) is a major protein in promoting the MMEJ pathway. In this study, we explored the effects of olaparib, a PARP inhibitor, in killing retinoblastoma cells. Retinoblastoma cell line Y79 and primary retinoblastoma cells expressed the cone-rod homeobox protein (CRX), a photoreceptor-specific marker. No detectable RB expression was found in these cells. The co-treatment of olaparib and etoposide led to enhanced cell death in both the Y79 cells and the primary retinoblastoma cells. Our results demonstrated the killing effects in retinoblastoma cells by PARP inhibitor olaparib after inducing DNA double-strand breaks. The use of olaparib in combination with etoposide could improve the cell-killing effects. Thus, lower dosages of etoposide can be used to treat retinoblastoma, which would potentially lead to a lower level of DSBs and a relatively more stable genome.

CRISPR-mediated host genomic DNA damage is efficiently repaired through microhomology-mediated end joining in Zymomonas mobilis

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against mobile genetic elements (MGEs) through uptake of invader-derived spacers. De novo adaptation samples spacers from both invaders and hosts, whereas primed adaptation shows higher specificity to sample spacers from invaders in many model systems as well as in the subtype I-F system of Zymomonas mobilis. Self-derived spacers will lead to CRISPR self-interference. However, our in vivo study demonstrated that this species used the microhomology-mediated end joining (MMEJ) pathway to efficiently repair subtype I-F CRISPR-Cas system-mediated DNA breaks guided by the self-targeting spacers. MMEJ repair of DNA breaks requires direct microhomologous sequences flanking the protospacers and leads to DNA deletions covering the protospacers. Importantly, CRISPR-mediated genomic DNA breaks failed to be repaired via MMEJ pathway in presence of higher copies of short homologous DNA. Moreover, CRISPR-cleaved exogenous plasmid DNA was failed to be repaired through MMEJ pathway, probably due to the inhibition of MMEJ by the presence of higher copies of the plasmid DNA in Z. mobilis. Our results infer that MMEJ pathway discriminates DNA damages between in the host chromosome versus mobile genetic element (MGE) DNA, and maintains genome stability post CRISPR immunity in Z. mobilis.

The Base Excision Repair Pathway in the Nematode Caenorhabditis elegans.

Exogenous and endogenous damage to the DNA is inevitable. Several DNA repair pathways including base excision, nucleotide excision, mismatch, homologous and non-homologous recombinations are conserved across all organisms to faithfully maintain the integrity of the genome. The base excision repair (BER) pathway functions to repair single-base DNA lesions and during the process creates the premutagenic apurinic/apyrimidinic (AP) sites. In this review, we discuss the components of the BER pathway in the nematode Caenorhabditis elegans and delineate the different phenotypes caused by the deletion or the knockdown of the respective DNA repair gene, as well as the implications. To date, two DNA glycosylases have been identified in C. elegans, the monofunctional uracil DNA glycosylase-1 (UNG-1) and the bifunctional endonuclease III-1 (NTH-1) with associated AP lyase activity. In addition, the animal possesses two AP endonucleases belonging to the exonuclease-3 and endonuclease IV families and in C. elegans these enzymes are called EXO-3 and APN-1, respectively. In mammalian cells, the DNA polymerase, Pol beta, that is required to reinsert the correct bases for DNA repair synthesis is not found in the genome of C. elegans and the evidence indicates that this role could be substituted by DNA polymerase theta (POLQ), which is known to perform a function in the microhomology-mediated end-joining pathway in human cells. The phenotypes observed by the C. elegans mutant strains of the BER pathway raised many challenging questions including the possibility that the DNA glycosylases may have broader functional roles, as discuss in this review.

Exploiting the Microhomology-Mediated End-Joining Pathway in Cancer Therapy.

Repair of DNA double-strand breaks (DSB) is performed by two major pathways, homology-dependent repair and classical nonhomologous end-joining. Recent studies have identified a third pathway, microhomology-mediated end-joining (MMEJ). MMEJ has similarities to homology-dependent repair, in that repair is initiated with end resection, leading to single-stranded 3' ends, which require microhomology upstream and downstream of the DSB. Importantly, the MMEJ pathway is commonly upregulated in cancers, especially in homologous recombination-deficient cancers, which display a distinctive mutational signature. Here, we review the molecular process of MMEJ as well as new targets and approaches exploiting the MMEJ pathway in cancer therapy.

Robust activation of microhomology-mediated end joining for precision gene editing applications.

One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.

Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

SummaryThe RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ∼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner.

Structure of the Helicase Domain of DNA Polymerase Theta Reveals a Possible Role in the Microhomology-Mediated End-Joining Pathway

SummaryDNA polymerase theta (Polθ) has been identified as a crucial alternative non-homologous end-joining factor in mammalian cells. Polθ is upregulated in a range of cancer cell types defective in homologous recombination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attractive target for cancer treatment. We present crystal structures of the helicase domain of human Polθ in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA-stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polθ exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polθ helicase domain in the context of the Polθ tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain.

Incidence of Genome Structure, DNA Asymmetry, and Cell Physiology on T-DNA Integration in Chromosomes of the Phytopathogenic Fungus Leptosphaeria maculans

The ever-increasing generation of sequence data is accompanied by unsatisfactory functional annotation, and complex genomes, such as those of plants and filamentous fungi, show a large number of genes with no predicted or known function. For functional annotation of unknown or hypothetical genes, the production of collections of mutants using Agrobacterium tumefaciens–mediated transformation (ATMT) associated with genotyping and phenotyping has gained wide acceptance. ATMT is also widely used to identify pathogenicity determinants in pathogenic fungi. A systematic analysis of T-DNA borders was performed in an ATMT-mutagenized collection of the phytopathogenic fungus Leptosphaeria maculans to evaluate the features of T-DNA integration in its particular transposable element-rich compartmentalized genome. A total of 318 T-DNA tags were recovered and analyzed for biases in chromosome and genic compartments, existence of CG/AT skews at the insertion site, and occurrence of microhomologies between the T-DNA left border (LB) and the target sequence. Functional annotation of targeted genes was done using the Gene Ontology annotation. The T-DNA integration mainly targeted gene-rich, transcriptionally active regions, and it favored biological processes consistent with the physiological status of a germinating spore. T-DNA integration was strongly biased toward regulatory regions, and mainly promoters. Consistent with the T-DNA intranuclear-targeting model, the density of T-DNA insertion correlated with CG skew near the transcription initiation site. The existence of microhomologies between promoter sequences and the T-DNA LB flanking sequence was also consistent with T-DNA integration to host DNA mediated by homologous recombination based on the microhomology-mediated end-joining pathway.

Microhomology-mediated deletion and gene conversion in African trypanosomes

Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in individual DSBR survivors exclusively revealed MMEJ-based deletions but no NHEJ. A survey of microhomologies typically revealed sequences of between 5 and 20 bp in length with several mismatches tolerated in longer stretches. Mean deletions were of 54 bp on the side closest to the break and 284 bp in total. Break proximity, microhomology length and GC-content all favored repair and the pattern of MMEJ described above was similar at several different loci across the genome. We also identified interchromosomal gene conversion involving HR and MMEJ at different ends of a duplicated sequence. While MMEJ-based deletions were RAD51-independent, one-sided MMEJ was RAD51 dependent. Thus, we describe the features of MMEJ in Trypanosoma brucei, which is analogous to micro single-strand annealing; and RAD51 dependent, one-sided MMEJ. We discuss the contribution of MMEJ pathways to genome evolution, subtelomere recombination and antigenic variation.