Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining.

In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects.

Disruption of DNA-PKcs-mediated cGAS retention on damaged chromatin potentiates DNA damage-inducing agent-induced anti-multiple myeloma activity.

BACKGROUND: Targeting DNA damage repair factors, such as DNA-dependent protein kinase catalytic subunit (DNA-PKcs), may offer an opportunity for effective treatment of multiple myeloma (MM). In combination with DNA damage-inducing agents, this strategy has been shown to improve chemotherapies partially via activation of cGAS-STING pathway by an elevated level of cytosolic DNA. However, as cGAS is primarily sequestered by chromatin in the nucleus, it remains unclear how cGAS is released from chromatin and translocated into the cytoplasm upon DNA damage, leading to cGAS-STING activation. METHODS: We examined the role of DNA-PKcs inhibition on cGAS-STING-mediated MM chemosensitivity by performing mass spectrometry and mechanism study. RESULTS: Here, we found DNA-PKcs inhibition potentiated DNA damage-inducing agent doxorubicin-induced anti-MM effect by activating cGAS-STING signaling. The cGAS-STING activation in MM cells caused cell death partly via IRF3-NOXA-BAK axis and induced M1 polarization of macrophages. Moreover, this activation was not caused by defective classical non-homologous end joining (c-NHEJ). Instead, upon DNA damage induced by doxorubicin, inhibition of DNA-PKcs promoted cGAS release from cytoplasmic chromatin fragments and increased the amount of cytosolic cGAS and DNA, activating cGAS-STING. CONCLUSIONS: Inhibition of DNA-PKcs could improve the efficacy of doxorubicin in treatment of MM by de-sequestrating cGAS in damaged chromatin.

Akt-mediated phosphorylation of XLF impairs non-homologous end-joining DNA repair.

Deficiency in repair of damaged DNA leads to genomic instability and is closely associated with tumorigenesis. Most DNA double-strand-breaks (DSBs) are repaired by two major mechanisms, homologous-recombination (HR) and non-homologous-end-joining (NHEJ). Although Akt has been reported to suppress HR, its role in NHEJ remains elusive. Here, we report that Akt phosphorylates XLF at Thr181 to trigger its dissociation from the DNA ligase IV/XRCC4 complex, and promotes its interaction with 14-3-3ß leading to XLF cytoplasmic retention, where cytosolic XLF is subsequently degraded by SCF(ß-TRCP) in a CKI-dependent manner. Physiologically, upon DNA damage, XLF-T181E expressing cells display impaired NHEJ and elevated cell death. Whereas a cancer-patient-derived XLF-R178Q mutant, deficient in XLF-T181 phosphorylation, exhibits an elevated tolerance of DNA damage. Together, our results reveal a pivotal role for Akt in suppressing NHEJ and highlight the tight connection between aberrant Akt hyper-activation and deficiency in timely DSB repair, leading to genomic instability and tumorigenesis.

ROS-Mediated Selective Killing Effect of Black Phosphorus: Mechanistic Understanding and Its Guidance for Safe Biomedical Applications.

Black phosphorus (BP)-based nanomaterials have distinguished advantages and potential applications in various biomedical fields. However, their biological effects in physiological systems remain largely unexplored. Here, we systematically revealed a reactive oxygen species (ROS)-mediated mechanism for the selective killing of cancer cells by BP-based nanosheets. The treatment with BP-based materials can induce higher levels of ROS in cancer cells than in normal cells, leading to significant changes in the cytoskeleton, cell cycle arrest, DNA damage, and apoptosis in tumor cell lines. We revealed that the decreased superoxide dismutase activity by lipid peroxides could be an essential mechanism of the selectively higher ROS generation induced by BP-based nanosheets in cancer cells. In addition, the selective killing effect only occurred within a certain dosage range (named "SK range" in this study). Once exceeding the SK range, BP-based materials could also induce a high ROS production in normal tissues, leading to detectable DNA damage and pathological characteristics in normal organs and raising safety concerns. These findings not only shed light on a new mechanism for the selective killing of cancer cells by BP-based materials but also provide deep insights into the safe use of BP-based therapies.

H2AX facilitates classical non-homologous end joining at the expense of limited nucleotide loss at repair junctions.

Phosphorylated histone H2AX, termed 'γH2AX', mediates the chromatin response to DNA double strand breaks (DSBs) in mammalian cells. H2AX deficiency increases the numbers of unrepaired DSBs and translocations, which are partly associated with defects in non-homologous end joining (NHEJ) and contributing to genomic instability in cancer. However, the role of γH2AX in NHEJ of general DSBs has yet to be clearly defined. Here, we showed that despite little effect on overall NHEJ efficiency, H2AX deficiency causes a surprising bias towards accurate NHEJ and shorter deletions in NHEJ products. By analyzing CRISPR/Cas9-induced NHEJ and by using a new reporter for mutagenic NHEJ, we found that γH2AX, along with its interacting protein MDC1, is required for efficient classical NHEJ (C-NHEJ) but with short deletions and insertions. Epistasis analysis revealed that ataxia telangiectasia mutated (ATM) and the chromatin remodeling complex Tip60/TRRAP/P400 are essential for this H2AX function. Taken together, these data suggest that a subset of DSBs may require γH2AX-mediated short-range nucleosome repositioning around the breaks to facilitate C-NHEJ with loss of a few extra nucleotides at NHEJ junctions. This may prevent outcomes such as non-repair and translocations, which are generally more destabilizing to genomes than short deletions and insertions from local NHEJ.

53BP1 Protects against CtIP-Dependent Capture of Ectopic Chromosomal Sequences at the Junction of Distant Double-Strand Breaks.

DNA double-strand breaks (DSB) are very harmful lesions that can generate genome rearrangements. In this study, we used intrachromosomal reporters to compare both the efficiency and accuracy of end-joining occurring with close (34 bp apart) vs. distant DSBs (3200 bp apart) in human fibroblasts. We showed that a few kb between two intrachromosomal I-SceI-induced DSBs are sufficient to foster deletions and capture/insertions at the junction scar. Captured sequences are mostly coupled to deletions and can be partial duplications of the reporter (i.e., sequences adjacent to the DSB) or insertions of ectopic chromosomal sequences (ECS). Interestingly, silencing 53BP1 stimulates capture/insertions with distant but not with close double-strand ends (DSEs), although deletions were stimulated in both case. This shows that 53BP1 protects both close and distant DSEs from degradation and that the association of unprotection with distance between DSEs favors ECS capture. Reciprocally, silencing CtIP lessens ECS capture both in control and 53BP1-depleted cells. We propose that close ends are immediately/rapidly tethered and ligated, whereas distant ends first require synapsis of the distant DSEs prior to ligation. This "spatio-temporal" gap gives time and space for CtIP to initiate DNA resection, suggesting an involvement of single-stranded DNA tails for ECS capture. We therefore speculate that the resulting single-stranded DNA copies ECS through microhomology-mediated template switching.

RAP80-directed tuning of BRCA1 homologous recombination function at ionizing radiation-induced nuclear foci.

In response to DNA double-strand breaks (DSBs), BRCA1 forms biochemically distinct complexes with certain other DNA damage response proteins. These structures, some of which are required for homologous recombination (HR)-type DSB repair, concentrate at distinct nuclear foci that demarcate sites of genome breakage. Polyubiquitin binding by one of these structures, the RAP80/BRCA1 complex, is required for efficient BRCA1 focal recruitment, but the relationship of this process to the execution of HR has been unclear. We found that this complex actively suppresses otherwise exaggerated, BRCA1-driven HR. By controlling the kinetics by which other BRCA1-interacting proteins that promote HR concentrate together with BRCA1 in nuclear foci, RAP80/BRCA1 complexes suppress excessive DSB end processing, HR-type DSB repair, and overt chromosomal instability. Since chromosomal instability emerges when BRCA1 HR function is either unbridled or absent, active tuning of BRCA1 activity, executed in nuclear foci, is important to genome integrity maintenance.

Buried territories: heterochromatic response to DNA double-strand breaks.

Cellular response to DNA double-strand breaks (DSBs), the most deleterious type of DNA damage, is highly influenced by higher-order chromatin structure in eukaryotic cells. Compared with euchromatin, the compacted structure of heterochromatin not only protects heterochromatic DNA from damage, but also adds an extra layer of control over the response to DSBs occurring in heterochromatin. One key step in this response is the decondensation of heterochromatin structure. This decondensation process facilitates the DNA damage signaling and promotes proper heterochromatic DSB repair, thus helping to prevent instability of heterochromatic regions of genomes. This review will focus on the functions of the ataxia telangiectasia mutated (ATM) signaling cascade involving ATM, heterochromatin protein 1 (HP1), Krüppel-associated box (KRAB)-associated protein-1 (KAP-1), tat-interacting protein 60 (Tip60), and many other protein factors in DSB-induced decondensation of heterochromatin and subsequent repair of heterochromatic DSBs. As some subsets of DSBs may be repaired in heterochromatin independently of the ATM signaling, a possible repair model is also proposed for ATM-independent repair of these heterochromatic DSBs.

Ataxia telangiectasia mutated (ATM) is dispensable for endonuclease I-SceI-induced homologous recombination in mouse embryonic stem cells.

Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency.

Double strand break repair functions of histone H2AX.

Chromosomal double strand breaks provoke an extensive reaction in neighboring chromatin, characterized by phosphorylation of histone H2AX on serine 139 of its C-terminal tail (to form "γH2AX"). The γH2AX response contributes to the repair of double strand breaks encountered in a variety of different contexts, including those induced by ionizing radiation, physiologically programmed breaks that characterize normal immune cell development and the pathological exposure of DNA ends triggered by telomere dysfunction. γH2AX also participates in the evolutionarily conserved process of sister chromatid recombination, a homologous recombination pathway involved in the suppression of genomic instability during DNA replication and directly implicated in tumor suppression. At a biochemical level, the γH2AX response provides a compelling example of how the "histone code" is adapted to the regulation of double strand break repair. Here, we review progress in research aimed at understanding how γH2AX contributes to double strand break repair in mammalian cells.

TREX2 enables efficient genome disruption mediated by paired CRISPR-Cas9 nickases that generate 3'-overhanging ends.

Paired SpCas9 nickases (SpCas9n) are an effective strategy to reduce off-target effect in genome editing. However, this approach is not efficient with 3'-overhanging ends, limiting its applications. In order to expand the utility of paired SpCas9n in genome editing, we tested the effect of the TREX2 3'-5' exonuclease on repair of 3'-overhanging ends. We found ectopic overexpression of Trex2 stimulates the efficiency of paired SpCas9n in genome disruption with 3'-overhanging ends up to 400-fold with little stimulation of off-target editing. TREX2 overexpressed preferentially deletes entire 3' overhangs but has no significant effect on 5' overhangs. Trex2 overexpression also stimulates genome disruption by paired SpCas9n that potentially generate short 3'-overhanging ends at overlapping SpCas9n target sites, suggesting sequential nicking of overlapping target sites by SpCas9n. This approach is further simplified with improved efficiency and safety by fusion of TREX2 and particularly its DNA-binding-deficient mutant to SpCas9n. Junction analysis at overlapping targets revealed the different extent of end resection of 3' single-stranded DNA (ssDNA) by free TREX2 and TREX2 fused to SpCas9n. SpCas9n-TREX2 fusion is more convenient and safer than overexpression of free TREX2 to process 3'-overhanging ends for efficient genome disruption by paired SpCas9n, allowing practical use of this TREX2-based strategy in genome editing.

Small extrachromosomal circular DNA harboring targeted tumor suppressor gene mutations supports intratumor heterogeneity in mouse liver cancer induced by multiplexed CRISPR/Cas9.

BACKGROUND: Primary liver cancer has significant intratumor genetic heterogeneity (IGH), which drives cancer evolution and prevents effective cancer treatment. CRISPR/Cas9-induced mouse liver cancer models can be used to elucidate how IGH is developed. However, as CRISPR/Cas9 could induce chromothripsis and extrachromosomal DNA in cells in addition to targeted mutations, we wondered whether this effect contributes to the development of IGH in CRISPR/Cas9-induced mouse liver cancer. METHODS: CRISPR/Cas9-based targeted somatic multiplex-mutagenesis was used to target 34 tumor suppressor genes (TSGs) for induction of primary liver tumors in mice. Target site mutations in tumor cells were analyzed and compared between single-cell clones and their subclones, between different time points of cell proliferation, and between parental clones and single-cell clones derived from mouse subcutaneous allografts. Genomic instability and generation of extrachromosomal circular DNA (eccDNA) was explored as a potential mechanism underlying the oscillation of target site mutations in these liver tumor cells. RESULTS: After efficiently inducing autochthonous liver tumors in mice within 30-60 days, analyses of CRISPR/Cas9-induced tumors and single-cell clones derived from tumor nodules revealed multiplexed and heterogeneous mutations at target sites. Many target sites frequently displayed more than two types of allelic variations with varying frequencies in single-cell clones, indicating increased copy number of these target sites. The types and frequencies of targeted TSG mutations continued to change at some target sites between single-cell clones and their subclones. Even the proliferation of a subclone in cell culture and in mouse subcutaneous graft altered the types and frequencies of targeted TSG mutations in the absence of continuing CRISPR/Cas9 genome editing, indicating a new source outside primary chromosomes for the development of IGH in these liver tumors. Karyotyping of tumor cells revealed genomic instability in these cells manifested by high levels of micronuclei and chromosomal aberrations including chromosomal fragments and chromosomal breaks. Sequencing analysis further demonstrated the generation of eccDNA harboring targeted TSG mutations in these tumor cells. CONCLUSIONS: Small eccDNAs carrying TSG mutations may serve as an important source supporting intratumor heterogeneity and tumor evolution in mouse liver cancer induced by multiplexed CRISPR/Cas9.

DNA nicks induce mutational signatures associated with BRCA1 deficiency.

Analysis of human cancer genome sequences has revealed specific mutational signatures associated with BRCA1-deficient tumors, but the underlying mechanisms remain poorly understood. Here, we show that one-ended DNA double strand breaks (DSBs) converted from CRISPR/Cas9-induced nicks by DNA replication, not two-ended DSBs, cause more characteristic chromosomal aberrations and micronuclei in Brca1-deficient cells than in wild-type cells. BRCA1 is required for efficient homologous recombination of these nick-converted DSBs and suppresses bias towards long tract gene conversion and tandem duplication (TD) mediated by two-round strand invasion in a replication strand asymmetry. However, aberrant repair of these nick-converted one-ended DSBs, not that of two-ended DSBs in Brca1-deficient cells, generates mutational signatures such as small indels with microhomology (MH) at the junctions, translocations and small MH-mediated TDs, resembling those in BRCA1-deficient tumors. These results suggest a major contribution of DNA nicks to mutational signatures associated with BRCA1 deficiency in cancer and the underlying mechanisms.

Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.

BACKGROUND: Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. RESULTS: In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. CONCLUSIONS: Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms, and holds tremendous promise in clinical applications. The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA (sgRNA) at a given site. However, little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks (DSBs). We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target, contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing. Here, we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing, and the opportunity this presents to optimize Cas9-based technology.

Quercetin Prevents Radiation-Induced Oral Mucositis by Upregulating BMI-1.

Radiation-induced oral mucositis is a major adverse event of radiotherapy. Severe oral mucositis may cause unwanted interruption in radiotherapy and reduce long-term survival in cancer patients receiving radiotherapy, but until now, there have been no effective options for preventing radiation-induced oral mucositis. Quercetin is a flavonoid that is widely found in food species and has anti-inflammatory, antioxidant, and anticancer activities. In this study, we investigated a new role of quercetin in preventing radiation-induced oral mucositis. Quercetin exerted preventive effects against radiation-induced oral mucositis induced by single-dose (25 Gy) ionizing radiation or fractionated ionizing radiation (8 Gy × 3) in C57BL/6 mice and maintained the proliferation ability of basal epithelial cells. Quercetin pretreatment alleviated reactive oxygen species generation, NF-κB pathway activation, and downstream proinflammatory cytokine production and reduced DNA double-strand breaks and cellular senescence induced by ionizing radiation. Quercetin also upregulated BMI-1 expression in oral epithelial cells and promoted ulcer repair. In addition, quercetin exerted similar radioprotective effects in irradiated primary cultured normal human keratinocytes, reduced reactive oxygen species generation and proinflammatory cytokine release, and promoted DNA double-strand break repair and wound healing by upregulating the expression of BMI-1, which is a polycomb group protein. Thus, quercetin can block multiple pathological processes of radiation-induced oral mucositis by targeting BMI-1 and may be a potential treatment option for preventing radiation-induced oral mucositis.

L ARP7 Is a BRCA1 Ubiquitinase Substrate and Regulates Genome Stability and Tumorigenesis.

Attenuated DNA repair leads to genomic instability and tumorigenesis. BRCA1/BARD1 are the best-known tumor suppressors that promote homology recombination (HR) and arrest cell cycle. However, it remains ambiguous whether and how their E3 ligase activity regulates HR. Here, we demonstrate that upon genotoxic stress, BRCA1 together with BARD1 catalyzes the K48 polyubiquitination on LARP7, a 7SK RNA binding protein known to control RNAPII pausing, and thereby degrades it through the 26S ubiquitin-proteasome pathway. Depleting LARP7 suppresses the expression of CDK1 complex, arrests the cell at the G2/M DNA damage checkpoint, and reduces BRCA2 phosphorylation, which thereby facilitates RAD51 recruitment to damaged DNA to enhance HR. Importantly, LARP7 depletion observed in breast cancer patients leads to chemoradiotherapy resistance both in vitro and in vivo. Altogether, this study unveils a mechanism by which BRCA1/BARD1 control HR and cell cycle, and highlights LARP7 as a potential target for cancer prevention and therapy.

Differential regulation of short- and long-tract gene conversion between sister chromatids by Rad51C.

The Rad51 paralog Rad51C has been implicated in the control of homologous recombination. To study the role of Rad51C in vivo in mammalian cells, we analyzed short-tract and long-tract gene conversion between sister chromatids in hamster Rad51C(-/-) CL-V4B cells in response to a site-specific chromosomal double-strand break. Gene conversion was inefficient in these cells and was specifically restored by expression of wild-type Rad51C. Surprisingly, gene conversions in CL-V4B cells were biased in favor of long-tract gene conversion, in comparison to controls expressing wild-type Rad51C. These long-tract events were not associated with crossing over between sister chromatids. Analysis of gene conversion tract lengths in CL-V4B cells lacking Rad51C revealed a bimodal frequency distribution, with almost all gene conversions being either less than 1 kb or greater than 3.2 kb in length. These results indicate that Rad51C plays a pivotal role in determining the "choice" between short- and long-tract gene conversion and in suppressing gene amplifications associated with sister chromatid recombination.

Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing.

BACKGROUND: Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. RESULTS: In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined "3n"-, "3n + 1"-, or "3n + 2"-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to "3n + 1"-bp, "3n + 2"-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. CONCLUSIONS: NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.

The mTOR-S6K pathway links growth signalling to DNA damage response by targeting RNF168.

Growth signals, such as extracellular nutrients and growth factors, have substantial effects on genome integrity; however, the direct underlying link remains unclear. Here, we show that the mechanistic target of rapamycin (mTOR)-ribosomal S6 kinase (S6K) pathway, a central regulator of growth signalling, phosphorylates RNF168 at Ser60 to inhibit its E3 ligase activity, accelerate its proteolysis and impair its function in the DNA damage response, leading to accumulated unrepaired DNA and genome instability. Moreover, loss of the tumour suppressor liver kinase B1 (LKB1; also known as STK11) hyperactivates mTOR complex 1 (mTORC1)-S6K signalling and decreases RNF168 expression, resulting in defects in the DNA damage response. Expression of a phospho-deficient RNF168-S60A mutant rescues the DNA damage repair defects and suppresses tumorigenesis caused by Lkb1 loss. These results reveal an important function of mTORC1-S6K signalling in the DNA damage response and suggest a general mechanism that connects cell growth signalling to genome stability control.