Error-prone Non-homologous End Joining Research Articles

DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline.

The male mammalian germline is characterized by substantial chromatin remodeling associated with the transition from histones to protamines during spermatogenesis, followed by the reversal to nucleohistones in the male pronucleus preceding the zygotic genome activation. Both transitions are associated with the extensive formation of DNA double-strand breaks (DSBs), requiring an estimated 5 to 10 million transient DSBs per spermatozoa. Additionally, the high transcription rate in early stages of spermatogenesis leads to transcription-coupled damage preceding meiotic homologous recombination, potentially further contributing to the DSB landscape in mature spermatozoa. Once meiosis is completed, spermatozoa remain haploid and therefore cannot rely on error-free homologous recombination, but instead depend on error-prone classical non-homologous end joining (cNHEJ). This DNA damage/repair-scenario is proposed to be one of the main causes of the observed paternal mutation propensity in human evolution. Recent studies have shown that DSBs in the male pronucleus are repaired by maternally provided Polθ in Caenorhabditis elegans through Polθ-mediated end joining (TMEJ). Additionally, population genetic datasets have revealed a preponderance of TMEJ signatures associated with human variation. Since these signatures are the result of the combined effect of TMEJ and DSB formation in spermatozoa and male pronuclei, we used a BLISS-based protocol to analyze recurrent DSBs in mature human sperm heads as a proxy of the male pronucleus before zygotic chromatin remodeling. The DSBs were found to be enriched in (YR)n short tandem repeats and in evolutionarily young SINEs, reminiscent to patterns observed in murine spermatids, indicating evolutionary hotspots of recurrent DSB formation in mammalian spermatozoa. Additionally, we detected a similar DSB pattern in diploid human IMR90 cells when cNHEJ was selectively inhibited, indicating the significant impact of absent cNHEJ on the sperm DSB landscape. Strikingly, regions associated with most retained histones, and therefore less condensed chromatin, were not strongly enriched with recurrent DSBs. In contrast, the fraction of retained H3K27me3 in the mature spermatozoa displayed a strong association with recurrent DSBs. DSBs in H3K27me3 are associated with a preference for TMEJ over cNHEJ during repair. We hypothesize that the retained H3K27me3 may trigger transgenerational DNA repair by priming maternal Polθ to these regions.

O-095 Assessment of genome editing outcomes in human preimplantation embryos subjected to CRISPR-Cas9 – most loss of heterozygosity (LOH) events are caused by DNA repair deficiency

Abstract Study question What is the cause of loss of heterozygosity, frequently observed in embryos subjected to CRISPR-Cas9, an interhomolog-homologous recombination repair (IH-HR) or deficiency in DNA repair? Summary answer Vast majority of LOH observed in human zygotes subjected to CRISPR-based editing is explained by deficiency in DNA repair prior to genome activation, not IH-HR. What is known already Genome editing (GE) at the preimplantation stage, intended to correct disease-causing mutations, is controversial due to safety and ethical concerns. It has been suggested that a high frequency of LOH seen in zygotes subjected to GE could be explained by resolution of the double-stranded DNA breaks (DSBs) created using CRISPR-Cas9 by interhomolog-homologous recombination repair (IH-HR), a process with potential to correct deleterious heterozygous mutations. However, such hypotheses are controversial and it is unclear whether embryos at this stage development can utilise IH-HR. CRISPR-Cas9 targeted sites are difficult to interrogate with traditional methods, risking a misinterpretation of editing outcomes. Study design, size, duration We hypothesised that the frequently observed LOH affecting the CRISPR-targeted sites in human zygotes is caused by the deficiency in DNA repair at this developmental stage, resulting in complex genotypes which traditional molecular methods have failed to detect, and not IH-HR which uses the second un-cleaved parental allele as a template for repair. DSBs were induced at three genomic sites in embryos microinjected at the time of fertilisation (n = 27) in this IRB-approved study. Participants/materials, setting, methods Research embryos were cultured for 60 hours, then disaggregated. Individual cells underwent an array of genetic tests, including NGS, allowing determination of the repair pathway utilised and revealing any cytogenetic abnormalities. A novel state-of-the-art analytical workflow was developed to interrogate 25kb around CRISPR cleavage sites. This approach provided a means of detecting complex genomic rearrangements as well as unresolved DNA damage, which would appear as LOH when employing traditional PCR-based approaches for assessment. Main results and the role of chance Induction of DSBs using CRISPR-Cas9 was 100% efficient when applied at the time of fertilisation. LOH was confirmed to be common at targeted sites, affecting 69/216 (32%) loci examined in 108 blastomeres. Remarkably, the great majority of LOH events (>90%) were a direct consequence of inappropriate/failed DNA repair (unresolved DNA breaks or complex genomic rearrangements). Strikingly, not a single instance of IH-HR was observed when CRISPR reagents were introduced at fertilisation. This may be explained by the physical separation of the two parental genomes in distinct pronuclei at that time. Allele-drop out (ADO), a technical problem, accounted for <10% of all LOH events. In total, 46% of DSBs failed to undergo appropriate repair, resulting in segmental aneuploidy (21%), large-scale deletions (10%), inversions/translocations (10%), or loss of the targeted chromosome (5%). 45% of DSBs were resolved using error-prone non-homologous end joining (NHEJ), while only 9% utilised the homology directed repair (HDR) pathway required for correction of mutations. Application of CRISPR-Cas9 to embryos on day-3 of development (after embryonic genome activation - EGA) was less efficient, only producing a DSB in 6/37 embryos (16%). However, the mode of repair differed markedly, 100% employing HDR (p = 0.0001). In 4 cases, this involved IH-HR. Limitations, reasons for caution This data provides strong evidence that LOH recorded in earlier studies was incorrectly attributed to IH-HR and was actually due to abnormal/failed repair. CRISPR appears more effective after EGA, with efficient DNA repair dominated by desirable HDR. However, a larger number of embryos is required to corroborate this finding. Wider implications of the findings The use of CRISPR-Cas9 at the time of fertilisation is frequently associated with problematic, unintended outcomes, rendering the technique clinically inapplicable in the current format. Careful study design which allows determination of all possible editing outcomes is essential and advances our understanding of the underlying mechanisms of DNA repair. Trial registration number not applicable

Introduction to CRISPR and Its Use in Drosophila.

The preeminence of Drosophila genetics has led to key discoveries in biology across a variety of fields and disciplines. The advent of CRISPR gene editing has expanded the toolkit of genetic reagents that can be applied to manipulate and observe genes, RNAs, and proteins in an in vivo context. This review describes CRISPR and its use as a transformative gene editing tool in Drosophila We focus on the canonical pathway in which the Cas9 nuclease is directed to specific sequences by guide RNA (gRNA), where cleavage leads to DNA repair by one of two main cellular pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The error-prone NHEJ pathway can be appropriated to disrupt targeted sequences, enabling a variety of loss-of-function studies. Induction of the HDR pathway allows precise editing, including defined deletions, the introduction of specific sequence changes, and the incorporation of fluorescent and epitope tags. These approaches have increased the power of Drosophila genetics and been successfully used to conduct in vivo structure-function studies, study disease-associated variants, and follow protein dynamics.

The cGAS-Ku80 complex regulates the balance between two end joining subpathways.

As the major DNA sensor that activates the STING-TBK1 signaling cascade, cGAS is mainly present in the cytosol. A number of recent reports have indicated that cGAS also plays critical roles in the nucleus. Our previous work demonstrated for the first time that cGAS is translocated to the nucleus upon the occurrence of DNA damage and inhibits homologous recombination (HR), one of the two major pathways of DNA double strand break (DSB) repair. However, whether nuclear cGAS regulates the other DSB repair pathway, nonhomologous end joining (NHEJ), which can be further divided into the less error-prone canonical NHEJ (c-NHEJ) and more mutagenic alternative NHEJ (alt-NHEJ) subpathways, has not been characterized. Here, we demonstrated that cGAS tipped the balance of the two NHEJ subpathways toward c-NHEJ. Mechanistically, the cGAS-Ku80 complex enhanced the interaction between DNA-PKcs and the deubiquitinase USP7 to improve DNA-PKcs protein stability, thereby promoting c-NHEJ. In contrast, the cGAS-Ku80 complex suppressed alt-NHEJ by directly binding to the promoter of Polθ to suppress its transcription. Together, these findings reveal a novel function of nuclear cGAS in regulating DSB repair, suggesting that the presence of cGAS in the nucleus is also important in the maintenance of genome integrity.

CDK9-55 guides the anaphase-promoting complex/cyclosome (APC/C) in choosing the DNA repair pathway choice.

DNA double-strand breaks (DSBs) contribute to genome instability, a key feature of cancer. DSBs are mainly repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). We investigated the role of an isoform of the multifunctional cyclin-dependent kinase 9, CDK9-55, in DNA repair, by generating CDK9-55-knockout HeLa clones (through CRISPR-Cas9), which showed potential HR dysfunction. A phosphoproteomic screening in these clones treated with camptothecin revealed that CDC23 (cell division cycle 23), a component of the E3-ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), is a new substrate of CDK9-55, with S588 being its putative phosphorylation site. Mutated non-phosphorylatable CDC23(S588A) affected the repair pathway choice by impairing HR and favouring error-prone NHEJ. This CDK9 role should be considered when designing CDK-inhibitor-based cancer therapies.

The dCas9-based genome editing in Plasmodium yoelii.

Genetic editing is a powerful tool for functional characterization of genes in various organisms. With its simplicity and specificity, the CRISPR-Cas9 technology has become a popular editing tool, which introduces site-specific DNA double-strand breaks (DSBs), and then leverages the endogenous repair pathway for DSB repair via homology-directed repair (HDR) or the more error-prone non-homologous end joining (NHEJ) pathways. However, in the Plasmodium parasites, the lack of a typical NHEJ pathway selects for DSB repair through the HDR pathway when a homologous DNA template is available. The AT-rich nature of the Plasmodium genome exacerbates this drawback by making it difficult to clone longer homologous repair DNA templates. To circumvent these challenges, we adopted the hybrid catalytically inactive Cas9 (dCas9)-microbial single-stranded annealing proteins (SSAP) editor to the Plasmodium genome. In Plasmodium yoelii, we demonstrated the use of the dCas9-SSAP, as the cleavage-free gene editor, by targeted gene deletion and gene tagging, even using shorter homologous DNA templates. This dCas9-SSAP method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, would be a great addition to the existing genetic toolbox and could be deployed for the functional characterization of genes in Plasmodium, contributing to improving the ability of the malaria research community in characterizing more than half of genes with unknown functions.IMPORTANCEMalaria caused by Plasmodium parasites infection remains a serious threat to human health, with an estimated 249 million malaria cases and 608,000 deaths worldwide in 2022, according to the latest report from the World Health Organization (WHO). Here, we demonstrated the use of dCas9-single-stranded annealing protein, as the cleavage-free gene editor in Plasmodium yoelii, by targeted deletion and gene tagging, even using shorter homologous DNA templates. This method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, showing the potential significance in greatly improving our ability to elucidate gene functions, would contribute to assisting the malaria research community in deciphering more than half of genes with unknown functions to identify new drug and vaccine targets.

Abstract B011: CDK9-55 guides the anaphase-promoting complex/cyclosome (APC/C) in choosing the DNA repair pathway by affecting the UFL1 ubiquitination

Abstract DNA double-strand breaks (DSBs) contribute to genome instability, a key feature of cancer. DSBs are mainly repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). We investigated the role of an isoform of the multifunctional cyclin-dependent kinase 9, CDK9-55, in DNA repair, by generating CDK9-55-knockout HeLa clones (through CRISPR-Cas9), which showed potential HR dysfunction. A phosphoproteomic screening in these clones treated with camptothecin (CPT) revealed that CDC23 (cell division cycle 23), a component of the E3-ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), is a new substrate of CDK9-55, with S588 being its putative phosphorylation site. Mutated non-phosphorylatable CDC23(S588A) affected the repair pathway choice by impairing HR and favoring error prone NHEJ. Moreover, CDC23(S588A) promoted the ubiquitination of UFL1, a recently identified HR player. Overall, CDK9-55 could guide APC/C in choosing the correct DNA repair pathway, possibly by regulating UFL1 stability. This CDK9 role should be considered when designing CDK-inhibitor-based cancer therapies. Citation Format: Luigi Alfano. CDK9-55 guides the anaphase-promoting complex/cyclosome (APC/C) in choosing the DNA repair pathway by affecting the UFL1 ubiquitination [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B011.

RPS19 and RPL5 Contribute to DNA Double-Strand Break Repair

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome most often due to germline pathogenic variants (PVs) in ribosomal protein (RP) genes, with RPS19 and RPL5 being the most frequently mutated. In addition to hypoplastic anemia, individuals have a moderately increased risk of myelodysplastic syndrome, acute myeloid leukemia, and solid cancers, most commonly colorectal cancer and osteosarcoma. A causal link between RP deficiency and cancer predisposition has yet to be firmly established. Our work aims to examine the impact of deficiencies in RPS19 and RPL5 on DNA double-strand break (DSB) repair with the overarching hypothesis that disruptions in DSB repair could increase mutagenesis and ultimately contribute to cancer development in DBA. We found that DBA patient-derived lymphoblastoid cells were hypersensitive to ionizing radiation (IR) and had delayed resolution of the DNA damage marker, γ-H2AX, at IR-induced foci. Additionally, we found that both RPS19- and RPL5-knocked down (KD) bone marrow-derived CD34+ cells had delayed resolution of DSBs after IR in neutral comet assays. These data suggest that RPS19 and RPL5 deficiencies lead to impaired DSB repair. We, therefore, assessed the effect of RPS19- and RPL5-KD on the four main DSB repair pathways using well-established U2OS (osteosarcoma) DSB reporter cell lines. We found decreased homologous recombination (HR) and single-strand annealing (SSA) upon RPS19-KD, whereas the efficiencies of nonhomologous end-joining (NHEJ) and alternative end-joining (alt-EJ) were unchanged. Consistent with an HR defect, RPS19-KD cells were hypersensitive to PARP inhibition. In contrast, RPL5-KD cells had increased NHEJ and alt-EJ, whereas the efficiencies of HR and SSA were unchanged. Together, these results suggest that shifts in DSB repair pathway choice, with less high-fidelity HR, or more error-prone NHEJ or mutagenic alt-EJ, could contribute to mutagenesis in the context of RPS19 or RPL5 PVs. To investigate the mechanism(s) underlying these effects, we examined cell cycle profiles and found decreased G1 and increased G2/M cells following both RPS19- and RPL5-KD, which suggests altered cell cycle profiles are not responsible for DSB repair effects since NHEJ occurs principally in G1 and HR is restricted to G2. Given the essential roles of RPS19 and RPL5 in translation, we also examined levels of crucial proteins in each DSB repair pathway. Surprisingly, we found increased levels of NHEJ proteins DNA ligase IV (LIG4) and XRCC4 in both RPS19 and RPL5-KD cells. However, restoring LIG4/XRCC4 to normal levels via siRNA KD did not normalize the DSB repair efficiencies. Since HR and SSA require extensive resection of the 5' DNA strand at DSBs, we examined whether RPS19-KD impaired this key step by assessing the accumulation of nuclear RPA2, which associates with end-resected DNA. We found nuclear RPA2 levels were not reduced but rather increased following IR compared to the control, indicating the reduction in HR and SSA efficiency in these cells was not due to impaired end resection and may, instead, involve a downstream step in HR that we are currently investigating. To assess if RPS19 and RPL5 might directly contribute to repair, we monitored the recruitment of fluorescently tagged RPS19 and RPL5 to DSBs. We found that these proteins were rapidly recruited to sites of laser microirradiation and sites of chromatin associated Fok1-nuclease (Figure 1), both of which induce DSBs. The recruitment of both proteins was hindered by PARP inhibition and enhanced for RPS19 with PARG inhibition, suggesting RPS19, in particular, may be parylated or interact with parylated proteins. Similar studies were performed in Saos2 cells, which, in contrast to U2OS cells, are p53 null. Whereas RPS19 was recruited to sites of laser microirradiation in Saos2 cells, RPL5 was not. Interestingly, a transcriptionally hyperactive p53 (∆CTD), but not a wild-type or a dominant-negative form of p53 (p.R248Q), restored RPL5 recruitment in Saos2 cells, suggesting a p53 downstream target is required for its recruitment. We are currently investigating proteins that mediate RPS19 and RPL5 DSB recruitment. Together, our results support a model whereby altered DNA DSB repair in RPS19- and RPL5-deficient cells may contribute to cancer development in DBA.

DNAR-12. ATR INHIBITION AS A NOVEL RADIOSENSITIZATION STRATEGY FOR GLIOBLASTOMA

Abstract Glioblastomas (GBM) are lethal brain tumors for which surgical resection followed by ionizing radiation (IR) with concurrent and adjuvant Temozolomide (TMZ) remains the mainstay of therapy. GBMs are extremely radioresistant, and radiosensitization approaches are desperately needed for these tumors. IR induces DNA double-strand breaks (DSBs), and these lesions can be repaired either by error-prone non-homologous end joining (NHEJ) or the error-free homologous recombination (HR) pathway. Research in our and other laboratories has shown that the ATR kinase promotes the HR pathway. We therefore hypothesized that ATR inhibition would block DSB repair in rapidly-dividing GBM cells that are HR dependent but not in non-dividing normal brain cells that are dependent upon NHEJ for repair. We treated a panel of GBM cell lines and normal human astrocytes with the ATR inhibitor VX-803 and/or IR and assessed for multiple endpoints including HR, DSB repair, and cell survival. We found that VX-803 blocks HR and DSB repair in GBM cells but does not significantly affect repair in non-dividing normal human astrocytes. Hence, treatment with VX-803 sensitizes GBM lines to IR in vitro as measured by the colony formation assay. Next, PDX mouse models of GBM were treated with VX-803 followed by XRT, and monitored for tumor growth and survival. We found that the drug can cross the blood-brain barrier and, in conjunction with IR, block DNA repair in intracranial tumors, attenuate tumor growth, and consequently extend survival of tumor-bearing mice. Importantly, the drug does not significantly affect DSB repair in normal brain cells indicating that ATR inhibition might selectively block repair in GBM cells, thereby minimizing normal tissue toxicity. Our results indicate that regulation of HR by ATR is critical for optimal DSB repair, especially in rapidly-dividing GBM cells, and that ATR inhibition could be used to improve GBM radiotherapy in the clinic.

Efficient repair of human genetic defect by CRISPR/Cas9-mediated interlocus gene conversion

Abstract DNA double-strand breaks (DSBs) induced by gene-editing tools are primarily repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR) using synthetic DNA templates. However, error-prone NHEJ may result in unexpected indels at the targeted site. For most genetic disorders, precise HDR correction using exogenous homologous sequence is ideal. But, the therapeutic application of HDR might be especially challenging given the requirement for the codelivery of exogenous DNA templates with toxicity into cells, and the low efficiency of HDR could also limit its clinical application. In this study, we efficiently repair pathogenic mutations in HBB coding regions of hematopoietic stem cells (HSCs) using CRISPR/Cas9-mediated gene conversion (CRISPR/GC) using the paralog gene HBD as the internal template. After transplantation, these edited HSCs successfully repopulate the hematopoietic system and generate erythroid cells with significantly reduced thalassemia propensity. Moreover, a range of pathogenic gene mutations causing β-thalassemia in HBB coding regions were effectively converted to normal wild-type sequences without exogenous DNA templates using CRISPR/GC. This highlights the promising potential of CRISPR/GC, independent of synthetic DNA templates, for genetic disease gene therapy.

Genetic control of the error-prone repair of a chromosomal double-strand break with 5' overhangs in yeast.

A targeted double-strand break introduced into the genome of Saccharomyces cerevisiae is repaired by the relatively error-prone nonhomologous end joining (NHEJ) pathway when homologous recombination is not an option. A zinc finger nuclease cleavage site was inserted out-of-frame into the LYS2 locus of a haploid yeast strain to study the genetic control of NHEJ when the ends contain 5' overhangs. Repair events that destroyed the cleavage site were identified either as Lys+ colonies on selective medium or as surviving colonies on rich medium. Junction sequences in Lys+ events solely reflected NHEJ and were influenced by the nuclease activity of Mre11 as well as by the presence/absence of the NHEJ-specific polymerase Pol4 and the translesion-synthesis DNA polymerases Pol ζ and Pol η. Although most NHEJ events were dependent on Pol4, a 29-bp deletion with endpoints in 3-bp repeats was an exception. The Pol4-independent deletion required translesion synthesis polymerases as well as the exonuclease activity of the replicative Pol δ DNA polymerase. Survivors were equally split between NHEJ events and 1.2 or 11.7 kb deletions that reflected microhomology-mediated end joining (MMEJ). MMEJ events required the processive resection activity of Exo1/Sgs1, but there unexpectedly was no dependence on the Rad1-Rad10 endonuclease for the removal of presumptive 3' tails. Finally, NHEJ was more efficient in nongrowing than in growing cells and was most efficient in G0 cells. These studies provide novel insights into the flexibility and complexity of error-prone DSB repair in yeast.

P38 MAPK inhibitor SB203580 enhances anticancer activity of PARP inhibitor olaparib in a synergistic way on non-small cell lung carcinoma A549 cells

The Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib gives promising results against various types of cancers in clinical trials. The combination of drugs always increases therapeutic efficacy because of targeting multiple pathways of cancer progression. Our objective was to explore the potential synergistic anticancer activities of olaparib combined with p38 MAPK inhibitor (MAPKi) SB203580 on non-small cell lung carcinoma (NSCLC) A549 cells. The effects of the individual compound and their combination on cell survival, DNA damage as detected by γH2AX foci, expression of key proteins in Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) repair, caspase 3 activation, nuclear fragmentation and telomerase regulation were studied in A549 cells. The results showed that olaparib and SB203580 individually reduced cell viability in a dose-dependent manner but combined treatment synergistically reduced cell viability. Olaparib combined with SB203580 significantly reduced error-free HR repair via reducing MRE11-RAD50 and promoted error-prone NHEJ repair by increasing Ku70-Ku80 leading to increased DNA damage-induced apoptosis. Notably, the alteration of proteins in HR/NHEJ pathways, DNA damage and induction of apoptosis was significant by combined treatment but not by 1 μM olaparib treatment alone. In addition, combined treatment reduced telomerase activity more than single treatment via reducing telomerase subunits. These data implicated that the anticancer potential of olaparib was significantly increased by combining SB203580 through increasing DNA damage-induced apoptosis and inhibiting telomerase activity.

Cryo-EM visualization of state transition during non-homologous end-joining (NHEJ).

DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors. Using single-particle cryo-electron microscopy, we visualized key DNA-protein complexes that are formed by human NHEJ factors. A near-complete NHEJ reaction cycle and a life cycle of DNA-PKcs (catalytic subunit of the DNA-dependent protein kinase) are proposed from the models. The Ku70/80 heterodimer (Ku), DNA-PKcs, XRCC4 and XLF first form a long-range synaptic complex in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, and XLF. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to substantial outward rotation of both Ku and DNA-PKcs to promote DNA double-strand break exposure and DNA-PKcs dissociation. Upon DNA-PKcs dissociation, the long-range synaptic complex transit into the short-range synaptic complex, within which the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, suggesting that the joining of both strands of a DSB involves both LigIV molecules. In addition, we captured a dimeric state of catalytically inactive DNA-PKcs, which resembles previously determined structures of PIKK family kinases, revealing a model of the full life cycle of DNA-PKcs during NHEJ.

Generation of genetically modified rat models via the CRISPR/Cas9 technology.

The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.

DYNLL1 transcript abundance decreases during oocyte and follicular growth and is associated with fertility in mice

DNA repair efficiency is an important determinant of oocyte quality and female fertility. DYNLL1 (Dynein light chain 1) plays a part in DNA double-strand break (DSB) repair choice by promoting error-prone non-homologous end joining (NHEJ) repair and in contrast by limiting accurate and error-free homologous recombination (HR) repair. Here, we showed that DYNLL1 transcript abundance decreases gradually during oocyte and follicular growth in mice. In parallel, DYNLL1 mRNA transcript levels decline in the course of oocyte meiotic maturation in mice. Furthermore, our in silico analysis showed that ovaries from high-fertility mice with increased ovulation have lower DYNLL1 transcript abundance compared with that of control mice. Similarly, we found that the induction of ovulation with human chorionic gonadotropin (hCG) decreases DYNLL1 mRNA transcript levels in cumulus oocyte complexes in mice. We hypothesize that this decrease in DYNLL1 transcript abundance during oocyte and follicular growth and in ovulation might lead to relatively higher error-free HR repair and lower error-prone NHEJ repair events in mice, resulting in increased genome stability. Increased genome stability in mice with lower DYNLL1 levels might also contribute to better fertility performance. This paper highlights the need for further in vivo and mechanistic studies to better understand the role of DYNLL1 down-regulation in the maintenance of oocyte reserve and in oocyte quality and fertility.

DNAR-14. Y-BOX BINDING PROTEIN 1 REGULATES MEDULLOBLASTOMA TUMOR BIOLOGY AND THERAPEUTIC RESPONSE

Abstract BACKGROUND Medulloblastoma (MB) is the most common pediatric brain tumor. MB can be dividing into 4 genetically distinct subgroups (Sonic Hedgehog (SHH), Wingless (WNT), Group 3, and Group 4). Most patients receive radiation and cisplatin backbone therapy. SHH and Group3 patients demonstrate poor prognosis, especially in cases of MYC amplification or p53 mutation. Given the continued reliance on DNA damage therapies, and our groups previous findings that YB1 regulates IGF2 transcription in SHH MB, we chose to investigate the role of YB1 in the DNA damage response and we performed RIPseq to understand mRNAs regulated by YB1 that may be targeted as novel therapeutics. METHODS Cell lines include primary MBCs derived from NeuroD2-SmoA1 SHH mice, ONS-76 human SHH, Daoy human SHH, D341 and D425 Human Group 3. YB1 overexpression or knockdown followed by radiation and assessment of yH2AX (a marker of DNA de-condensation following damage), Comet assay (a marker of physical damage), and proliferation time courses were used to assess effects of YB1 modulation on radiation response. RIPseq and YB1 knockdown were used to assess the effects of YB1 on PLXND1 levels. Scratch assay and western blotting of EMT markers were used to assess effects of PLXND1 silencing on migration. RESULTS (Radiation Studies) Overexpression of YB1 in primary MBCs followed by allograft into BL6 mice results in decreased overall survival. YB1 silencing followed by radiation results in faster resolution of yH2AX, faster resolution of damage, and lack of pRPA32 accumulation. YB1 silencing sensitizes cells to radiation resulting in substantial decreases in proliferation. (RNA Binding Protein Studies) YB1 binds and positively regulates PLXND1 translation. Silencing PLXND1 results in decreased migration and EMT mark expression. CONCLUSIONS YB1 drives a more error prone non-homologous End-Joining based mechanism of repair and binds and regulates PLXND1 post-transcriptionally. PLXND1 drives a migratory phenotype in SHH MB.

Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells.

Repair of double-strand breaks (DSBs) in somatic cells is primarily accomplished by error-prone nonhomologous end joining and less frequently by precise homology-directed repair preferentially using the sister chromatid as a template. Here, a Drosophila system performs efficient somatic repair of both DSBs and single-strand breaks (SSBs) using intact sequences from the homologous chromosome in a process we refer to as homologous chromosome-templated repair (HTR). Unexpectedly, HTR-mediated allelic conversion at the white locus was more efficient (40 to 65%) in response to SSBs induced by Cas9-derived nickases D10A or H840A than to DSBs induced by fully active Cas9 (20 to 30%). Repair phenotypes elicited by Nickase versus Cas9 differ in both developmental timing (late versus early stages, respectively) and the production of undesired mutagenic events (rare versus frequent). Nickase-mediated HTR represents an efficient and unanticipated mechanism for allelic correction, with far-reaching potential applications in the field of gene editing.

Double-Stranded Break Repair in Mammalian Cells and Precise Genome Editing.

In mammalian cells, double-strand breaks (DSBs) are repaired predominantly by error-prone non-homologous end joining (NHEJ), but less prevalently by error-free template-dependent homologous recombination (HR). DSB repair pathway selection is the bedrock for genome editing. NHEJ results in random mutations when repairing DSB, while HR induces high-fidelity sequence-specific variations, but with an undesirable low efficiency. In this review, we first discuss the latest insights into the action mode of NHEJ and HR in a panoramic view. We then propose the future direction of genome editing by virtue of these advancements. We suggest that by switching NHEJ to HR, full fidelity genome editing and robust gene knock-in could be enabled. We also envision that RNA molecules could be repurposed by RNA-templated DSB repair to mediate precise genetic editing.

Recent Advances in the Development of Non-PIKKs Targeting Small Molecule Inhibitors of DNA Double-Strand Break Repair.

The vast majority of cancer patients receive DNA-damaging drugs or ionizing radiation (IR) during their course of treatment, yet the efficacy of these therapies is tempered by DNA repair and DNA damage response (DDR) pathways. Aberrations in DNA repair and the DDR are observed in many cancer subtypes and can promote de novo carcinogenesis, genomic instability, and ensuing resistance to current cancer therapy. Additionally, stalled or collapsed DNA replication forks present a unique challenge to the double-strand DNA break (DSB) repair system. Of the various inducible DNA lesions, DSBs are the most lethal and thus desirable in the setting of cancer treatment. In mammalian cells, DSBs are typically repaired by the error prone non-homologous end joining pathway (NHEJ) or the high-fidelity homology directed repair (HDR) pathway. Targeting DSB repair pathways using small molecular inhibitors offers a promising mechanism to synergize DNA-damaging drugs and IR while selective inhibition of the NHEJ pathway can induce synthetic lethality in HDR-deficient cancer subtypes. Selective inhibitors of the NHEJ pathway and alternative DSB-repair pathways may also see future use in precision genome editing to direct repair of resulting DSBs created by the HDR pathway. In this review, we highlight the recent advances in the development of inhibitors of the non-phosphatidylinositol 3-kinase-related kinases (non-PIKKs) members of the NHEJ, HDR and minor backup SSA and alt-NHEJ DSB-repair pathways. The inhibitors described within this review target the non-PIKKs mediators of DSB repair including Ku70/80, Artemis, DNA Ligase IV, XRCC4, MRN complex, RPA, RAD51, RAD52, ERCC1-XPF, helicases, and DNA polymerase θ. While the DDR PIKKs remain intensely pursued as therapeutic targets, small molecule inhibition of non-PIKKs represents an emerging opportunity in drug discovery that offers considerable potential to impact cancer treatment.

Nuclear and mitochondrial DNA editing in human cells with zinc finger deaminases

Base editing in nuclear DNA and mitochondrial DNA (mtDNA) is broadly useful for biomedical research, medicine, and biotechnology. Here, we present a base editing platform, termed zinc finger deaminases (ZFDs), composed of custom-designed zinc-finger DNA-binding proteins, the split interbacterial toxin deaminase DddAtox, and a uracil glycosylase inhibitor (UGI), which catalyze targeted C-to-T base conversions without inducing unwanted small insertions and deletions (indels) in human cells. We assemble plasmids encoding ZFDs using publicly available zinc finger resources to achieve base editing at frequencies of up to 60% in nuclear DNA and 30% in mtDNA. Because ZFDs, unlike CRISPR-derived base editors, do not cleave DNA to yield single- or double-strand breaks, no unwanted indels caused by error-prone non-homologous end joining are produced at target sites. Furthermore, recombinant ZFD proteins, expressed in and purified from E. coli, penetrate cultured human cells spontaneously to induce targeted base conversions, demonstrating the proof-of-principle of gene-free gene therapy.