Targeted DNA Double-strand Breaks Research Articles

684P - Targeting c-met and DNA double-strand break (DSB) repair pathways for BRCA-mutated gastric carcinomas

Background: In the present study, we underline the importance of combining c-Met Inhibitor (Inh.) and DSB repair Inhibitor (PARP Inh.) with the goal of accomplishing a synergistic therapeutic strategy in BRCA-mutated gastric carcinomas. Methods: Firstly, to verify whether c-Met affects tumor response to PARP Inh., AGS (low c-Met expression) and Hs746T (high c-Met expression) cell lines, were subjected to knockdown c-Met expression, in the presence of PARP Inh. (NU1025). Subsequently, we examined the correlation between BRCA1/2 and c-Met. We knocked down BRCA1/2 expression in AGS and Hs746T cells and treated them with PARP Inh. Next, we examined the effects of combining c-Met Inh. (SU11274) and PARP Inh. (NU1025), measured by MTT, clonogenic cell survival and Annexin, assays. We also evaluated the effect of combining PARP Inh. and c-Met Inh. in AGS/Hs746T xenograft tumor models. AGS/Hs746T -pcDNA3 and AGS/Hs746T -siBRCA cells were injected into SCID mice. Tumor sizes were measured every 3 days. DNA damage was assessed by γ-H2AX staining. Results: Silence of Met expression promoted Hs746T cells more sensitive to PARP Inh. to similar levels as the AGS cells, as indicated by decreased cell viability. Silence of BRCA1/2 expression sensitized only AGS cells, signifying that increased expression of c-Met renders cells resistant to PARP Inh. in the context of inactivating BRCA1/2. Combining c-Met Inh. and PARP Inh. suppressed cell growth and clonogenicity and enhanced apoptosis in both AGS and Hs746T cells. The dual inhibition was demonstrated to be even more successful when cells were knockdown for BRCA1/2. Also, co-inhibition treatment substantially reduced tumor growth to AGS/Hs746T -pcDNA3 and more even effectively to AGS/Hs746T –siBRCA1/2 xenograft models, compared to either Inh. alone. DNA damage was higher in AGS/Hs746T –siBRCA1/2 compare to AGS/Hs746T -pcDNA3 xenograft models. Conclusions: BRCA deficiency renders gastric tumor cells sensitive to PARP inhibition. In addition, treatment with c-Met Inh. enhanced Hs746T sensitivity to the PARP Inh. Our data demonstrate that dual Met/PARP inhibition is synergistic providing an effective therapeutic strategy in BRCA-mutated gastric carcinomas. Legal entity responsible for the study: M.V. Karamouzis Funding: None Disclosure: All authors have declared no conflicts of interest.

Abstract PR19: Targeting DNA double-strand break repair to potentiate radio- and chemo-therapy of glioblastoma

Abstract Glioblastomas (GBMs) are lethal brain tumors which are treated with ionizing radiation (IR) in combination with the DNA alkylating agent temozolomide (TMZ). Unfortunately, not all GBMs respond to therapy, and most of them quickly acquire resistance to TMZ and recur. In order to better understand the molecular basis for therapy-driven TMZ resistance, mice bearing orthotopic GBM xenografts were subjected to protracted TMZ treatment, and cell lines were generated from the primary (untreated) and recurrent (TMZ-treated) tumors. We found that cell lines derived from recurrent tumors were more resistant to TMZ in vitro compared to cell lines derived from primary tumors. We also found that the increased resistance to TMZ was due to augmented repair of TMZ-induced DNA double strand breaks (DSBs). TMZ induces DNA replication-associated DSBs that are primarily repaired by the homologous recombination (HR) pathway. Augmented HR appears to underpin TMZ resistance in the recurrent lines as these cells were cross-resistant to other agents that induce replication-associated DSBs, exhibited faster resolution of damage-induced Rad51 foci, and displayed higher levels of sister chromatid exchanges (SCEs). We have recently shown that CDKs 1 and 2 promote HR in S and G2 phases of the cell cycle, in part, by phosphorylating the exonuclease EXO1. We hypothesized, therefore, that blocking CDKs 1 and 2 might be a viable strategy for re-sensitizing recurrent tumors to TMZ. Indeed, we found that CDK inhibitors, AZD5438 and Roscovitine, could attenuate HR in the recurrent TMZ-resistant cell lines, resulting in significant chemo-sensitization. While TMZ-induced DSBs are primarily repaired by HR, IR- induced DSBs are mainly repaired by the non-homologous end joining (NHEJ) pathway. We, therefore, developed another approach to sensitize GBMs to both radiation and TMZ by using a dual PI3K/mTOR inhibitor, NVP-BEZ235, to target both DNA-PKcs and ATM, key enzymes in the NHEJ and HR pathways, respectively. We found that NVP-BEZ235 inhibited both DNA-PKcs and ATM in orthotopic GBM xenografts, thus blocking the repair of TMZ- and IR-induced DSBs in these tumors. Hence NVP-BEZ235, administered with IR, could attenuate tumor growth, and extend survival of tumor-bearing mice. Importantly, inhibition of DSB repair was more pronounced in tumor cells compared to normal brain cells, thereby providing a larger therapeutic window. In sum, these studies indicate that augmented DSB repair may underlie radio- and chemo-resistance in GBM, and provide support for DNA repair inhibition as an effective strategy for improving the efficacy of GBM therapy. This abstract is also being presented as Poster B22. Citation Format: Bipasha Mukherjee, Carlos Gil del Alcazar, Pavlina Todorova, Sandeep Burma. Targeting DNA double-strand break repair to potentiate radio- and chemo-therapy of glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR19.

Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes.

Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR–Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9–gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species.

Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

Abstract B20: In silico and in vitro drug screening identifies new therapeutic approaches targeting DNA double-strand breaks repair in Ewing sarcoma

Abstract Purpose: Ewing sarcoma (EWS) is the second most frequent malignant bone and soft-tissue tumor in children and young adults. More than 85% of EWS expressed the oncogenic transcription factor EWS-FLI1 fusion protein. With current therapies, about 75% of patients with localized disease survive, but patients with metastatic or recurrent disease have a dismal outcome with survival rates less than 20%. Although significant advances have been made in understanding the molecular basis of EWS, little has changed in the treatment of EWS patients over the past four decades. Efforts have been focused on the development of therapeutics targeting the EWS-FLI1 oncoprotein; however, no effective drugs have been developed. Therefore, our goal was to accelerate promising and new combination therapies to EWS patients through drug repurposing and rediscovery, and to expand the treatment options and improve treatment outcomes for patients with this rare and life-threatening disease. Methods: We first employed an integrated gene expression signatures-based bioinformatics approach to identify FDA-approved drugs that are predicted to reverse the EWS disease gene signatures and/or mimic the silencing of the EWS-FLI1 oncogene. We then evaluated the drug hits from the in silico approaches in three benign sarcoma cell lines (Hs 919.T., Hs 822.T., Hs 863.T.) and a panel of six genetically diverse EWS cell lines (A673, TC71, SK-ES-1, RD-ES, CHLA-258 and COG-E-352) by using CellTiter-Blue® Cell Viability Assay. The cytotoxic activities and drug interaction of the most potent drugs in various combinations were further assessed in vitro by using 8x8 matrix dose response assay. The combination index (CI) was calculated by the CompuSyn software according to the Chou and Talalay's method. Cellular apoptosis and DNA damage response after drug treatments in EWS cells were analyzed by Western blot. Results: Twenty-six FDA-approved drugs were identified through in silico prediction approaches (FDR < 0.05). Among them, MS-275, elesclomol, daunorubicin and vindesine are the most potent and selective drugs in killing EWS cells (72 h, IC50 0.004 - 3.65 µM), as compare to the benign sarcoma cells (IC50s in EWS cells were at least 10 times lower than the counterparts in benign controls). The combination of MS-275 (Class I HDAC inhibitor) and elesclomol (a selective mitochondrial reactive oxygen species inducer) was the most robust and synergistic treatment in inhibiting EWS cell growth, with 57%-82% of the combinations showing synergy (CI < 1) in the matrix dose response test across all tested EWS cell lines with either EWS-FLI1 or EWS-ERG fusion. Western blot data indicated that MS-275 and elesclomol induced caspase-dependent apoptosis in EWS cells in both time- and dose-dependent manner. Both MS-275 and elesclomol treatment led to DNA double-strand breaks (DSBs), as evidenced by the phosphorylation of γH2AX. MS-275 treatment inhibited homologous recombination (HR) pathway by downregulating BRCA1 and RAD51, while MS-275 regulated non-homologous end-joining (NHEJ) pathway in a cell type-dependent manner. Conclusions: Our studies revealed that the combination of MS-275 and elesclomol were synergistic and that MS-275 sensitized EWS cells to elesclomol through targeting DNA DSBs repair. This combination therapy may provide an alternative treatment strategy for children and young adults diagnosed with Ewing sarcoma. Citation Format: Yan Ma, Bin Chen, Scott J. Weir, Atul Butte, Andrew K. Godwin. In silico and in vitro drug screening identifies new therapeutic approaches targeting DNA double-strand breaks repair in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B20.

Gene targeting and transgene stacking using intra genomic homologous recombination in plants.

Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform.

High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

SummaryCRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

High-frequency, precise modification of the tomato genome.

BackgroundThe use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.ResultsHere, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences.ConclusionsHigh-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0796-9) contains supplementary material, which is available to authorized users.

Preventing Damage Limitation: Targeting DNA-PKcs and DNA Double-Strand Break Repair Pathways for Ovarian Cancer Therapy.

Platinum-based chemotherapy is the cornerstone of ovarian cancer treatment, and its efficacy is dependent on the generation of DNA damage, with subsequent induction of apoptosis. Inappropriate or aberrant activation of the DNA damage response network is associated with resistance to platinum, and defects in DNA repair pathways play critical roles in determining patient response to chemotherapy. In ovarian cancer, tumor cell defects in homologous recombination – a repair pathway activated in response to double-strand DNA breaks (DSB) – are most commonly associated with platinum-sensitive disease. However, despite initial sensitivity, the emergence of resistance is frequent. Here, we review strategies for directly interfering with DNA repair pathways, with particular focus on direct inhibition of non-homologous end joining (NHEJ), another DSB repair pathway. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a core component of NHEJ and it has shown considerable promise as a chemosensitization target in numerous cancer types, including ovarian cancer where it functions to promote platinum-induced survival signaling, via AKT activation. The development of pharmacological inhibitors of DNA-PKcs is on-going, and clinic-ready agents offer real hope to patients with chemoresistant disease.

New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double-strand breaks. Targeting of the DNA break is typically controlled by a single-guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR-Cas9 technology can be used for efficient, marker-free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time-consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9-sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter-selection using 5-fluoro-orotic acid (5-FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR-Cas9 technology in yeast genome editing.

Transgenic trait deployment using designed nucleases.

The demand for crops requiring increasingly complex combinations of transgenes poses unique challenges for transgenic trait deployment. Future value-adding traits such as those associated with crop performance are expected to involve multiple transgenes. Random integration of transgenes not only results in unpredictable expression and potential unwanted side effects but stacking multiple, randomly integrated, independently segregating transgenes creates breeding challenges during introgression and product development. Designed nucleases enable the creation of targeted DNA double-strand breaks at specified genomic locations whereby repair can result in targeted transgene integration leading to precise alterations in DNA sequences for plant genome editing, including the targeting of a transgene to a genomic locus that supports high-level and stable transgene expression without interfering with resident gene function. In addition, targeted DNA integration via designed nucleases allows for the addition of transgenes into previously integrated transgenic loci to create stacked products. The currently reported frequencies of independently generated transgenic events obtained with site-specific transgene integration without the aid of selection for targeting are very low. A modular, positive selection-based gene targeting strategy has been developed involving cassette exchange of selectable marker genes which allows for targeted events to be preferentially selected, over multiple cycles of sequential transformation. This, combined with the demonstration of intragenomic recombination following crossing of transgenic events that contain stably integrated donor and target DNA constructs with nuclease-expressing plants, points towards the future of trait stacking that is less dependent on high-efficiency transformation.

Targeting DNA Double-strand Break Repair in Cancer Therapy

Stephanie C Her1 and Chengtao Her2* 1Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, USA 2School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Washington, USA *Corresponding author: Chengtao Her, School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA, Tel: 5093357537; E-mail: cher@wsu.edu

Genome Editing in Erythroid Progenitor Cells Mediated By Crispr/Cas9

Investigation of the genetic underpinnings of erythrocyte development holds great value not only in the development of potential therapeutics for hematologic disorders, but also for elucidating basic biological principles. A robust model system for studying erythropoiesis is the mouse fetal liver system, as murine fetal liver is predominantly composed of erythroid progenitor cells at 2 weeks gestation. Upon isolation, these cells can be cultured in the presence of erythropoietic cytokines and follow distinct phases of development, from immature erythroid progenitors to terminally differentiated erythrocytes with robust enucleation and hemoglobinization.To date, loss of function genetic studies of erythropoiesis using the mouse fetal liver system have relied on mouse strains deficient in a gene of interest, or RNA interference inhibiting translation of a gene product of interest. Both strategies have limitations in terms of either time-intensive generation of genetically deficient mice, or inability of RNA interference to faithfully model homozygous deficiency, or haploinsufficiency. The development of genome editing technology based on a RNA-guided system for inducing targeted DNA double strand breaks (DSBs) raises the possibility of faithfully modeling homozygous deficiency or haploinsufficiency in a significantly higher throughput manner. This system consists of RNA-based Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) elements complexed with the Cas9 nuclease. Upon expression of both components in eukaryotic cells, a CRISPR single guide RNA (sgRNA), base pairs with a genomic target, guiding Cas9 to induce a DSB at that site. Genome editing then occurs at the site of the break if repair occurs via the non-homologous end joining DNA repair pathway, which produces mutational insertions, deletions, and substitutions during the process of DSB repair.In this study, we aimed to develop a system for CRISPR/Cas9-mediated genome editing in murine fetal liver cells. We constructed a retroviral vector co-expressing the Cas9 nuclease and an sgRNA. We initially designed sgRNAs targeting 2 genes non-essential for erythroid development, Gata3, which encodes a transcription factor required for T-cell development, and Lcp2, which encodes an adapter protein required for signal transduction during T-cell activation. These genes were chosen in order to assay genome editing efficiency without the occurrence of negative selection against disruption of genes required for erythroid development. Transduction of fetal liver cells isolated on embryonic day 14.5 (E14.5) with a retroviral vector expressing Cas9 and an sgRNA targeting Gata3 resulted in editing of 38% of Gata3 alleles. Transduction of E14.5 fetal liver cells with vector targeting Lcp2 resulted in editing of 15% of Lcp2 alleles. No editing was detected in control cells transduced with a retroviral vector expressing Cas9 and a scrambled sgRNA. Genome editing was detected using the Surveyor nuclease assay, which quantifies allelic frequency of gene mutations resulting from DSB repair by non-homologous end joining. We next designed an sgRNA targeting the Bcl11a gene, which encodes a protein shown to be instrumental in the embryonic to adult globin switch in mice. Transduction of E14.5 fetal liver cells with vector targeting Bcl11a resulted in editing of 49% of Bcl11a alleles. We then assessed if constitutive expression of Cas9 and an sgRNA affects the ability of fetal liver cells to undergo terminal erythroid differentiation. Compared to cells transduced with vector expressing only GFP, fetal liver cells transduced with retroviral vectors expressing Cas9 and scrambled sgRNAs had no significant difference in enucleation rate, a marker of terminal erythroid differentiation.In this study we demonstrate the ability to induce robust levels of genome editing at various genomic sites in mouse fetal liver cells using CRISPR/Cas9. We also demonstrate constitutive expression of Cas9 does not have any detrimental effect on enucleation. These results open the possibility of high-throughput modeling of homozygous genetic deficiency and genetic haploinsufficiency in studies of erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector.

Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers. We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells. We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codon-swapping, leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60% activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Off-target analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs. These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFN-mediated genome editing of primary cells, for both ex vivo or in vivo applications.

Every silver lining has a cloud: the scientific and animal welfare issues surrounding a new approach to the production of transgenic animals.

The scientific basis and advantages of using recently developed CRISPR/Cas-9 technology for transgenesis have been assessed with respect to other production methods, laboratory animal welfare, and the scientific relevance of transgenic models of human diseases in general. As the new technology is straightforward, causes targeted DNA double strand breaks and can result in homozygous changes in a single step, it is more accurate and more efficient than other production methods and speeds up transgenesis. CRISPR/Cas-9 also obviates the use of embryonic stem cells, and is being used to generate transgenic non-human primates (NHPs). While the use of this method reduces the level of animal wastage resulting from the production of each new strain, any long-term contribution to reduction will be offset by the overall increase in the numbers of transgenic animals likely to result from its widespread usage. Likewise, the contribution to refinement of using a more-precise technique, thereby minimising the occurrence of unwanted genetic effects, will be countered by a probable substantial increase in the production of transgenic strains of increasingly sentient species. For ethical and welfare reasons, we believe that the generation of transgenic NHPs should be allowed only in extremely exceptional circumstances. In addition, we present information, which, on both welfare and scientific grounds, leads us to question the current policy of generating ever-more new transgenic models in light of the general failure of many of them, after over two decades of ubiquitous use, to result in significant advances in the understanding and treatment of many key human diseases. Because this unsatisfactory situation is likely to be due to inherent, as well as possibly avoidable, limitations in the transgenic approach to studying disease, which are briefly reviewed, it is concluded that a thorough reappraisal of the rationale for using genetically-altered animals in fundamental research and by the pharmaceutical industry, and for its support by funding bodies, should be undertaken. In the meantime, the use of CRISPR/Cas-9 to generate new transgenic cells in culture is to be guardedly encouraged.

Toward establishing an efficient and versatile gene targeting system in higher plants

Abstract Precise modification of plant genomes provides powerful tools for understanding gene function. Furthermore, pinpoint modification of endogenous plant genes is also an attractive method in molecular plant breeding. One of the best methods currently available to induce specific DNA sequence changes into genomes is gene targeting (GT) via homologous recombination (HR). GT can induce a variety of mutations, including substitution of several nucleotides as well as insertions and deletions. Of the various approaches taken to improve GT efficiency, utilization of engineered sequence-specific endonucleases to create targeted DNA double-strand breaks (DSBs) that stimulate HR at breaking sites is one of the most effective. As a universal selection system for GT cells, a target-gene-independent selection system such as positive–negative selection with subsequent elimination of the positive selection marker using piggyBac transposon or HR combined with mega-nuclease allows retention of the desired mutation without any trace of additional exogenous nucleotides. Furthermore, a novel GT system named in planta GT seems to have effectively overcome problems due to low transformation efficiency of the HR template into plant nuclei, and can adjust the timing of DSB induction at the target locus. In the in planta system, an HR template flanked by a nuclease recognition site is stably integrated into the plant genome; subsequent expression of nuclease simultaneously releases the HR template from the genome and induces a DSB at the target site. Here, we review recent developments in GT, giving examples of new techniques improving the efficiency of GT in plants.

Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks

The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks.

SiRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/− with susceptibility versus C57BL/6-Trp53+/− mice with resistance to mammary tumors

BALB/c mice heterozygous for Trp53 develop a high proportion of spontaneous mammary tumors, a phenotype distinct from other mouse strains. BALB/c-Trp53+/− female mice, thus, resemble the hereditary Li-Fraumeni syndrome (LFS) characterized by early-onset of breast cancer, even though LFS involves TP53 mutations, which may involve not only loss- but also gain-of-function. Previous analysis of tumors in BALB/c-Trp53+/− females showed frequent loss of heterozygosity involving the wild-type allele of Trp53 and displayed characteristics indicative of mitotic recombination. Critical involvement of DNA double-strand break (DSB) repair dysfunction, particularly of homologous recombination (HR), was also noticed in the etiology of human breast cancer. To better define functional alterations in BALB/c-Trp53+/− mice, we applied a fluorescence-based DSB repair assay on mouse embryonic fibroblasts (MEFs) from BALB/c-Trp53+/− versus C57BL/6J-Trp53+/− mice. This approach revealed deregulation of HR but not non-homologous end-joining (NHEJ) in BALB/c-Trp53+/−, which was further confirmed for mammary epithelial cells. Screening of a small interfering RNA-library targeting DSB repair, recombination, replication and signaling genes, identified 25 genes causing differences between homologous DSB repair in the two strains upon silencing. Interactome analysis of the hits revealed clustering of replication-related and fanconi anemia (FA)/breast cancer susceptibility (BRCA) genes. Further dissection of the functional change in BALB/c-Trp53+/− by immunofluorescence microscopy of nuclear 53BP1, Replication protein A (RPA) and Rad51 foci uncovered differences in crosslink and replication-associated repair. Chromosome breakage, G2 arrest and biochemical analyses indicated a FA pathway defect downstream of FancD2 associated with reduced levels of BRCA2. Consistent with polygenic models for BRCA, mammary carcinogenesis in BALB/c-Trp53+/− mice may, therefore, be promoted by a BRCA modifier allele in the FA pathway in the context of partial p53 loss-of-function.

Quantitative assay for TALEN activity at endogenous genomic loci

SummaryArtificially designed nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can induce a targeted DNA double-strand break at the specific target genomic locus, leading to the frameshift-mediated gene disruption. However, the assays for their activity on the endogenous genomic loci remain limited. Herein, we describe a versatile modified lacZ assay to detect frameshifts in the nuclease target site. Short fragments of the genome DNA at the target or putative off-target loci were amplified from the genomic DNA of TALEN-treated or control embryos, and were inserted into the lacZα sequence for the conventional blue–white selection. The frequency of the frameshifts in the fragment can be estimated from the numbers of blue and white colonies. Insertions and/or deletions were easily determined by sequencing the plasmid DNAs recovered from the positive colonies. Our technique should offer broad application to the artificial nucleases for genome editing in various types of model organisms.

High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes

Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells.