Homology-directed Repair Mechanisms Research Articles

BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.

Abstract B019: BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response

Abstract Alternative Lengthening of Telomeres (ALT) is a homology-directed repair (HDR) mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. ALT cells exhibit more endogenous replication stress and DNA damage at the telomere compared to ALT-negative cells. The Bloom Syndrome helicase (BLM) is critical for ALT and promotes replication stress and DNA damage specifically at telomeres in ALT cells. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication stress-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2 commensurate with the appearance of telomere C-strand specific single-stranded DNA. Moreover, DNA2 nuclease deficiency increased 5’-flap formation in a BLM-dependent manner, and BLM promoted ALT in response to telomere C-strand, but not G-strand, nicks. These findings define the seminal events in the ALT DNA damage response, linking aberrant lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers (Jiang et al. submitted). These findings will be discussed and how BLM activity is a key determinant of the specific vulnerability of ALT cells to FANCM inhibition, which is currently being developed to target ALT-dependent cancers. Citation Format: Haoyang Jiang, Tianpeng Zhang, Hardeep Kaur, Tao Shi, Aravind Krishnan, Youngho Kwon, Patrick Sung, Roger Greenberg. BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response [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 B019.

Distinct modes of telomere synthesis and extension contribute to Alternative Lengthening of Telomeres

Alternative lengthening of telomeres (ALT) is a homology-directed repair mechanism that becomes activated in a subset of cancers to maintain telomere length. One of the defining features of ALT cells is the prevalence of extrachromosomal telomeric repeat (ECTR) DNA. Here, we identify that ALT cells engage in two modes of telomere synthesis. Non-productive telomere synthesis occurs during the G2 phase of the cell cycle and is characterized by newly synthesized internal telomeric regions that are not retained in the subsequent G1, coinciding with an induction of ECTR DNA. Productive telomere synthesis occurs specifically during the transition from G2 to mitosis and is defined as the extension of the telomere termini. While many proteins associated with break-induced telomere synthesis function in both non-productive and productive telomere synthesis, POLH specifically promotes productive telomere lengthening and suppresses non-productive telomere synthesis. These findings delineate the mechanism and cell cycle regulation of ALT-mediated telomere synthesis and extension.

Noodles, the all-in-one system for on-target efficiency analysis of CRISPR guide RNAs

The efficiency of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (gRNA) targeting is critical for CRISPR associated protein 9 (Cas9)-dependent genomic modifications. Here, we developed Noodles, an all-in-one system to test the on-target activity of gRNAs easily and efficiently. Single-strand annealing repair mechanism of the split luciferase gene allows a positive selection of gRNAs efficiently driving nuclease activity of Cas9 from Streptococcus pyogenes (SpCas9). Our system can reliably validate in silico-predicted gRNAs before implementing them for in vitro and in vivo applications. Altogether, Noodles might be an asset for researchers and bioengineers, saving their time and efforts, while keeping the screening efficient and sensitive.•All-in-one dual-luciferase system to easily probe on-target activity of gRNAs based on homology-directed repair mechanism.•Easy-to-subclone spCas9-based 2-plasmid system comprising Renilla luciferase for transfection efficiency control.

DNAR-07. MOLECULAR BIOMARKERS ASSOCIATED WITH TARGETED DISRUPTION OF ALTERNATIVE LENGTHENING OF TELOMERES IN ATRX-DEFICIENT GLIOMA MODELS

Abstract Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism of telomere maintenance that occurs in approximately 10-15 percent of all cancers. ALT enables the replicative immortality of cancer cells and is characterized by elevated levels of DNA replication stress and telomere-localized DNA double-strand breaks (DSBs). In gliomas, ALT frequently co-occurs with inactivating mutations in ATRX, which is thought to promote ALT induction by disrupting ATRX-mediated histone H3.3 deposition at telomeres, thus leading to telomere synthesis by homology-directed repair mechanisms. To identify potential therapeutic vulnerabilities specific to ALT-positive tumors, we identified candidate ALT-positive cancer cell lines in the Cancer Dependency Map (DepMap) dataset and validated the presence or absence of ALT phenotypic markers, including ALT-associated PML bodies (APBs) and nuclear foci of single-stranded telomeric DNA (ssTeloC). Comparing ALT-positive cells to all other cancer cell lines in the DepMap dataset, we identified the replication fork remodeling enzymes SMARCAL1 and FANCM as gene dependencies with the highest degree of ALT-specificity. To validate SMARCAL1 as an ALT-specific dependency, we used inducible RNAi to deplete SMARCAL1 in ALT-positive glioma cells. SMARCAL1 depletion caused a marked increase in the abundance and intensity of ssTeloC nuclear foci, as well as increased γH2AX abundance in ALT+ glioma cells but not telomerase-positive cells. Inducible SMARCAL1 depletion did not coincide with detectable increases in apoptotic markers (e.g. caspase-3 cleavage). However, SMARCAL1 depletion caused a significant increase in the abundance of nuclear abnormalities indicative of mitotic catastrophe (e.g. micronuclei, nucleoplasmic bridges, and fragmented/multilobular nuclei). In an orthotopic model of ALT-positive glioma, doxycycline-inducible RNAi targeting SMARCAL1 extended the median survival of tumor-bearing animals to 155 days compared to 111 days in the non-targeting control condition. These data validate the therapeutic targetability of SMARCAL1 in ALT-positive gliomas and support the development of small molecule SMARCAL1 inhibitors for treatment of ALT-positive tumors.

Break-induced replication orchestrates resection-dependent template switching.

Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres1,2. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear3-5. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments(PICh) to capture the telomeric DNA damage response proteome during BITS1,6. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.

OPA1 Dominant Optic Atrophy: Pathogenesis and Therapeutic Targets.

OPA1 Dominant Optic Atrophy: Pathogenesis and Therapeutic Targets.

Cytoplasmic Injection of Zygotes to Genome Edit Naturally Occurring Sequence Variants Into Bovine Embryos.

Genome editing provides opportunities to improve current cattle breeding strategies through targeted introduction of natural sequence variants, accelerating genetic gain. This can be achieved by harnessing homology-directed repair mechanisms following editor-induced cleavage of the genome in the presence of a repair template. Introducing the genome editors into zygotes and editing in embryos has the advantage of uncompromised development into live animals and alignment with contemporary embryo-based improvement practices. In our study, we investigated the potential to introduce sequence variants, known from the pre-melanosomal protein 17 (PMEL) and prolactin receptor (PRLR) genes, and produce non-mosaic, edited embryos, completely converted into the precision genotype. Injection of gRNA/Cas9 editors into bovine zygotes to introduce a 3 bp deletion variant into the PMEL gene produced up to 11% fully converted embryos. The conversion rate was increased to up to 48% with the use of TALEN but only when delivered by plasmid. Testing three gRNA/Cas9 editors in the context of several known PRLR sequence variants, different repair template designs and delivery as DNA, RNA or ribonucleoprotein achieved full conversion rates up to 8%. Furthermore, we developed a biopsy-based screening strategy for non-mosaic embryos which has the potential for exclusively producing non-mosaic animals with intended precision edits.

Development of transgenic Daphnia magna for visualizing homology-directed repair of DNA

In the crustacean Daphnia magna, studying homology-directed repair (HDR) is important to understand genome maintenance during parthenogenesis, effects of environmental toxicants on the genome, and improvement of HDR-mediated genome editing. Here we developed a transgenic D. magna that expresses green fluorescence protein (GFP) upon HDR occurrence. We utilized the previously established reporter plasmid named DR-GFP that has a mutated eGFP gene (SceGFP) and the tandemly located donor GFP gene fragment (iGFP). Upon double-strand break (DSB) introduction on SceGFP, the iGFP gene fragment acts as the HDR template and restores functional eGFP expression. We customized this reporter plasmid to allow bicistronic expression of the mCherry gene under the control of the D. magna EF1α-1 promoter/enhancer. By CRISPR/Cas-mediated knock-in of this plasmid via non-homologous joining, we generated the transgenic D. magna that expresses mCherry ubiquitously, suggesting that the DR-GFP reporter gene is expressed in most cells. Introducing DSB on the SceGFP resulted in eGFP expression and this HDR event could be detected by fluorescence, genomic PCR, and quantitative reverse-transcription PCR, suggesting this line could be used for evaluating HDR. The established reporter line might expand our understanding of the HDR mechanism and also improve the HDR-based gene-editing system in this species.

Anti-recombination function of MutSα restricts telomere extension by ALT-associated homology-directed repair.

SUMMARYAlternative lengthening of telomeres (ALT) is a telomere-elongation mechanism observed in ~15% of cancer subtypes. Current models indicate that ALT is mediated by homology-directed repair mechanisms. By disrupting MSH6 gene expression, we show that the deficiency of MutSα (MSH2/MSH6) DNA mismatch repair complex causes striking telomere hyperextension. Mechanistically, we show MutSα is specifically recruited to telomeres in ALT cells by associating with the proliferating-cell nuclear antigen (PCNA) subunit of the ALT telomere replisome. We also provide evidence that MutSα counteracts Bloom (BLM) helicase, which adopts a crucial role in stabilizing hyper-extended telomeres and maintaining the survival of MutSα-deficient ALT cancer cells. Lastly, we propose a model in which MutSα deficiency impairs heteroduplex rejection, leading to premature initiation of telomere DNA synthesis that coincides with an accumulation of telomere variant repeats (TVRs). These findings provide evidence that the MutSα DNA mismatch repair complex acts to restrain unwarranted ALT.

Effects of RAD51-stimulatory compound 1 (RS-1) and its vehicle, DMSO, on pig embryo culture

Pigs have become an important model for agricultural and biomedical purposes. The advent of genomic engineering tools, such as the CRISPR/Cas9 system, has facilitated the production of livestock models with desired modifications. However, precise site-specific modifications in pigs through the homology-directed repair (HDR) pathway remains a challenge. In mammalian embryos, the use of small molecules to inhibit non-homologous end joining (NHEJ) or to improve HDR have been tested, but little is known about their toxicity. The compound RS-1 stimulates the activity of the RAD51 protein, which plays a key role in the HDR mechanism, demonstrating enhancement of HDR events in rabbit and bovine zygotes. Thus, in this study, we evaluated the dosage and temporal effects of RS-1 on porcine embryo development and viability. Additionally, we assessed the effects of its vehicle, DMSO, during embryo in vitro culture. Transient exposure to 7.5 μM of RS-1 did not adversely affect early embryo development and was compatible with subsequent development to term. Additionally, low concentrations of its vehicle, DMSO, did not show any toxicity to in vitro produced embryos. The transient use of RS-1 at 7.5 μM during in vitro culture seems to be the best protocol of choice to reduce the potentially toxic effects of RS-1 while attempting to improve HDR in the pig. Direct injection of the CRISPR/Cas9 system, combined with strategies to increase the frequency of targeted modifications via HDR, have become an important tool to simplify and accelerate the production of genetically modified livestock models.

Multiplex Genome Editing in Yeast by CRISPR/Cas9 - A Potent and Agile Tool to Reconstruct Complex Metabolic Pathways.

Numerous important pharmaceuticals and nutraceuticals originate from plant specialized metabolites, most of which are synthesized via complex biosynthetic pathways. The elucidation of these pathways is critical for the applicable uses of these compounds. Although the rapid progress of the omics technology has revolutionized the identification of candidate genes involved in these pathways, the functional characterization of these genes remains a major bottleneck. Baker’s yeast (Saccharomyces cerevisiae) has been used as a microbial platform for characterizing newly discovered metabolic genes in plant specialized metabolism. Using yeast for the investigation of numerous plant enzymes is a streamlined process because of yeast’s efficient transformation, limited endogenous specialized metabolism, partially sharing its primary metabolism with plants, and its capability of post-translational modification. Despite these advantages, reconstructing complex plant biosynthetic pathways in yeast can be time intensive. Since its discovery, CRISPR/Cas9 has greatly stimulated metabolic engineering in yeast. Yeast is a popular system for genome editing due to its efficient homology-directed repair mechanism, which allows precise integration of heterologous genes into its genome. One practical use of CRISPR/Cas9 in yeast is multiplex genome editing aimed at reconstructing complex metabolic pathways. This system has the capability of integrating multiple genes of interest in a single transformation, simplifying the reconstruction of complex pathways. As plant specialized metabolites usually have complex multigene biosynthetic pathways, the multiplex CRISPR/Cas9 system in yeast is suited well for functional genomics research in plant specialized metabolism. Here, we review the most advanced methods to achieve efficient multiplex CRISPR/Cas9 editing in yeast. We will also discuss how this powerful tool has been applied to benefit the study of plant specialized metabolism.

Mapping CRISPR-Cas9 public and commercial innovation using The\xa0Lens institutional toolkit

CRISPR-Cas9 is a revolutionary technology because it is precise, fast and easy to implement, cheap and components are readily accessible. This versatility means that the technology can deliver a timely end product and can be used by many stakeholders. In plant cells, the technology can be applied to knockout genes by using CRISPR–Cas nucleases that can alter coding gene regions or regulatory elements, alter precisely a genome by base editing to delete or regulate gene expression, edit precisely a genome by homology-directed repair mechanism (cellular DNA), or regulate transcriptional machinery by using dead Cas proteins to recruit regulators to the promoter region of a gene. All these applications can be for: 1) Research use (Non commercial), 2) Uses related product components for the technology itself (reagents, equipment, toolkits, vectors etc), and 3) Uses related to the development and sale of derived end products based on this technology. In this contribution, we present a prototype report that can engage the community in open, inclusive and collaborative innovation mapping. Using the open data at the Lens.org platform and other relevant sources, we tracked, analyzed, organized, and assembled contextual and bridged patent and scholarly knowledge about CRISPR-Cas9 and with the assistance of a new Lens institutional capability, The Lens Report Builder, currently in beta release, mapped the public and commercial innovation pathways of the technology. When scaled, this capability will also enable coordinated editing and curation by credentialed experts to inform policy makers, businesses and private or public investment.

92 Correction of the CFTR G542X mutation using CRISPR/Cas9 genome editing in ovine-bovine interspecies embryos

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have recently generated 3 CF sheep models: a CFTR−/− model (Fan et al. 2018 LCI Insight 3:e123529; https://doi.org/10.1172/jci.insight.123529) and 2 additional models where we introduced human G542X and F508del mutations into the sheep genome (unpublished). Correction of CFTR mutations in zygotes with gene-editing techniques could be a permanent solution to cure this disease. To assess the efficiency of mutation correction invitro by CRISPR/Cas9, we utilised embryos generated by ovine-bovine interspecies SCNT (iSCNT) due to limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos, in which nucleus donors were derived from ovine fibroblasts and recipient cytoplasm from enucleated bovine oocytes. These iSCNT embryos were able to develop to 16- to 32-cell stage (3/30, 10.0%), which allowed the genotyping of each embryo using PCR-restriction fragment length polymorphism assays and Sanger sequencing. Then, specific single-guide RNAs (sgRNAs) and 101-bp single-stranded oligodeoxynucleotides (ssODNs) were designed and synthesised to correct the G542X mutation in the sheep CFTR gene. We optimized the concentrations of Cas9:sgRNA ribonucleoproteins (RNPs) for 1-cell stage embryonic injection. Mutation analysis of embryos was conducted at 3 days post injection. Genotyping results showed that we achieved high efficiencies (95.7–100%) of mutations (indels) at targeting loci after injection of different concentrations of Cas9:sgRNA RNPs (0.02 µg:0.6 pmol/µL to 1.4 µg:40 pmol/µL). Furthermore, when an RNP (1.4 µg:40 pmol/µL) was co-injected with a ssODN (80 pmol/µL), both targeting the G542X mutation, the mutation was successfully corrected in the genome of iSCNT embryos generated using G542X fibroblasts as nucleus donors at an efficiency of 5.7% (3/53) via homology-directed repair mechanism. During the invitro culture of iSCNT embryos, we did not observe significant difference (P>0.05, unpaired t-test) in cleavage rates between embryos with or without injection (85.5% vs. 89.0%). Off-target analysis of those mutated and G542X-corrected embryos is in progress. Our strategy overcomes the limitation of oocyte source and provides an opportunity to mimic the editing of any other gene in embryos of different species.

Detection of CRISPR-mediated genome modifications through altered methylation patterns of CpG islands

BackgroundThe development and application of CRISPR technologies for the modification of the genome are rapidly expanding. Advances in the field describe new CRISPR components that are strategically engineered to improve the precision and reliability of CRISPR editing within the genome sequence. Genome modification using induced genome breaks that are targeted and mediated by CRISPR components leverage cellular mechanisms for repair like homology directed repair (HDR) to incorporate genomic edits with increased precision.ResultsIn this report, we describe the gain of methylation at typically hypomethylated CpG island (CGI) locations affected by the CRISPR-mediated incorporation of donor DNA using HDR mechanisms. With characterization of CpG methylation patterns using whole genome bisulfite sequencing, these CGI methylation disruptions trace the insertion of the donor DNA during the genomic edit. These insertions mediated by homology-directed recombination disrupt the generational methylation pattern stability of the edited CGI within the cells and their cellular lineage within the animal strain, persisting across generations. Our approach describes a statistically based workflow for indicating locations of modified CGIs and provides a mechanism for evaluating the directed modification of the methylome of the affected CGI at the CpG-level.ConclusionsWith advances in genome modification technology comes the need to detect the level and persistence of methylation change that modifications to the genomic sequence impose upon the collaterally edited methylome. Any modification of the methylome of somatic or germline cells could have implications for gene regulation mechanisms governed by the methylation patterns of CGI regions in the application of therapeutic edits of more sensitively regulated genomic regions. The method described here locates the directed modification of the mouse epigenome that persists over generations. While this observance would require supporting molecular observations such as direct sequence changes or gene expression changes, the observation of epigenetic modification provides an indicator that intentionally directed genomic edits can lead to collateral, unintentional epigenomic changes post modification with generational persistence.

Development of gene editing strategies for human β-globin (HBB) gene mutations

Recent developments in gene editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene editing tools are promising candidates for clinical applications, especially for treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in human β-globin gene (HBB). Clinical strategies have demonstrated substantial success, however there is not any permanent cure for SCD available. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (gRNA) to induce sequence-specific DNA double strand break (DSB). When this accompanies a repair template, it allows repairing the mutated gene. In this study, it was aimed to target HBB gene via CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in human cell line, by Homology Directed Repair (HDR). We have achieved to induce target specific nucleotide changes on HBB gene in the locus of mutation causing SCD. The effect of on-target activity of bone fide standard gRNA and newly developed longer gRNA were examined. It is observed that longer gRNA has higher affinity to target DNA while having the same performance for targeting and Cas9 induced DSBs. HDR mechanism was triggered by co-delivery of donor DNA repair templates in circular plasmid form. In conclusion, we have suggested methodological pipeline for efficient targeting with higher affinity to target DNA and generating desired modifications on HBB gene.

Characterization of a Genetically Engineered HUDEP2 Cell Line Harboring a Sickle Cell Disease Mutation As a Potential Research Tool for Preclinical Sickle Cell Disease Drug Discovery

Sickle cell disease (SCD) is a genetic disorder caused by a missense mutation in the beta-globin gene [Glu6Val] resulting in the formation of HbS (sickle hemoglobin). HbS polymerizes under low oxygen tension causing an array of severe pathophysiological complications, including vaso-occlusion, pain and hemolytic anemia. Traditionally, research efforts toward studying anti-sickling effects of various pathways and compounds in vitro have been made using sickle cell patient derived samples. However, such studies are limited based on sample availability and a large degree of phenotypic variability. Thus, a cultured cell system with a sickle mutation mimicking human sickle cell erythroid cells is a potentially useful tool that can be used to interrogate anti-sickling compound action and to study various facets of sickle cell phenotype. In this study, we generated a genetically engineered sickle mutation (GAG to GTG) in HUDEP2 cell line using CRISPR Cas9 gene editing and homology directed repair mechanism. HUDEP2 cells are immortalized human erythroid progenitor cells, obtained from the Riken Institute and were cultured as described (Kurita et al 2013). CRISPR Cas9 RNP complex with modified guide RNA along with ssODN were electroporated in HUDEP2 cells. Cells were also treated with SCR7, a NHEJ inhibitor to facilitate homology- directed repair events. Cells were sorted for clonal selection, cultured and further screened for homozygous knock-in mutations using digital droplet PCR. The mutation was further confirmed by sanger sequencing. 12 clones consisting of homozygous mutation were identified, with 6 of these clones selected for further characterization. Cells were differentiated for 7 days in culture and hemolysates were analyzed using HPLC. Heterozygote clone showed 32.3% HbS which was comparable to sickle cell trait patient derived blood samples whereas homozygous clones (ssHUDEP2) showed between 71-85% HbS. These results were further confirmed for the presence of HbS using mass spectrometry. Mutant HUDEP2 cell growth and differentiation were similar to the wild-type HUDEP2 cells. To evaluate sickling in these cells we optimized the differentiation protocol to improve the maturation of these cells by withdrawing SCF and doxycycline gradually. Hemoglobin polymerization and resulting shape change in sickle HUDEP cells was determined by subjecting mature ssHUDEP2 cells to hypoxia (1% oxygen) for 3 hours. Upon subjecting to hypoxia, ssHUDEP2 cells showed morphological changes indicative of HbS polymerization. These morphological changes were absent in wild type cells under similar conditions of hypoxia. In addition, ssHUDEP2 cells pre-treated with 25µM GBT440, a known anti-sickling compound, for 1 hour prior to hypoxia incubation elicited a reduction in the shape change associated with hypoxia, suggesting inhibition of hemoglobin polymerization and sickling. These results suggest that genetically-engineered sickle mutant HUDEP cells represent a preclinical model for development of anti-sickling compounds for the potential treatment of Sickle Cell Disease and to explore novel mechanistic pathways. Disclosures Gupta: Sanofi: Employment. Sturtevant:Sanofi: Employment. Vieira:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. Demers:Sanofi: Employment.

Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

A gene drive biases the transmission of one of two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems were recently developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR/Cas9 and endogenous homology directed repair mechanisms to convert heterozygous genotypes to homozygosity1–4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (e.g., to model multigenic human diseases). However, such a system has not yet been demonstrated in mammals. Here, we utilize an “active genetic” element that encodes a guide RNA embedded in the mouse Tyrosinase gene to evaluate whether targeted gene conversion can occur when CRISPR/Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double strand DNA breaks in the early embryo and male germline, these breaks are not resolved by homology directed repair. However, Cas9 expression limited to the female germline forms double strand breaks that are resolved by homology directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate feasibility of CRISPR/Cas9-mediated systems that bias inheritance in mice, which have potential to transform the use of rodent models in basic and biomedical research.

Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair.

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.

How to Generate Non-Mosaic CRISPR/Cas9 Mediated Knock-In and Mutations in F0 Xenopus Through the Host-Transfer Technique.

We have taken advantage of the well-established oocyte host transfer technique to optimize a method for CRISPR editing of Xenopus that provides an efficient non-mosaic targeted insertion of small DNA fragment through homology-directed repair mechanism.