Homology-directed Repair Rates Research Articles

Increasing Knockin Efficiency in Mouse Zygotes by Transient Hypothermia.

Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates. Using a traffic light reporter in mouse embryonic stem cells and a fluorescence conversion reporter in human-induced pluripotent stem cells, we confirm that a transient cold shock leads to an increase in the rate of HDR, with a corresponding decrease in the rate of NHEJ repair. We then investigated whether a similar cold shock could lead to an increase in the rate of HDR in the mouse embryo. By analyzing the efficiency of gene editing using single nucleotide polymorphism changes and loxP insertion at three different genetic loci, we found that a transient reduction in temperature after zygote electroporation of CRISPR-Cas9 ribonucleoprotein with a single-stranded oligodeoxynucleotide repair template did indeed increase knockin efficiency, without affecting embryonic development. The efficiency of gene editing with and without the cold shock was first assessed by genotyping blastocysts. As a proof of concept, we then confirmed that the modified embryo culture conditions were compatible with live births by targeting the coat color gene tyrosinase and observing the repair of the albino mutation. Taken together, our data suggest that a transient cold shock could offer a simple and robust way to improve knockin outcomes in both stem cells and zygotes.

445 Awardee Talk: Generation of a BMPR2 Heterozygous Ovine Genetic Model for Heritable Pulmonary Arterial Hypertension

Abstract Pulmonary Arterial Hypertension (PAH) is a rare vascular disorder affecting 1 to 2 cases per million individuals. Monoallelic mutations in the BMPR2 gene are the most common genetic risk factor, with about 20% of carriers affected. BMPR2 is a serine/threonine kinase receptor in the transforming growth factor beta (TGF-β) superfamily. Despite well-known associations with PAH, the role of BMPR2 in the progression of the disorder remains poorly understood due in part to the lack of appropriate preclinical genetic models. The homozygous BMPR2 (-/-) knockout genotype is embryonic lethal, which is an obstacle to creating a suitable genetic biomedical model for PAH. To overcome this barrier, we used a single guide RNA (sgRNA) CRISPR/Cas9 approach to target exon 3 of the ovine BMPR2 gene, in combination with a single-stranded oligo-deoxynucleotide (ssODN) synonymous homology-directed repair (HDR) template to create heterozygous BMPR2 (+/-) sheep. Heterozygote lambs could be generated in two ways with this approach – high editing efficiency coupled with ssODN homology-directed repair of one of the edited alleles, OR intermediate editing efficiency with low to no presence of HDR in the genetic sequence. Our main objectives were to:1.) Determine optimal concentrations of ssODN (100, 300, and 600 ng/ul) to promote HDR and 2.) Identify the sgRNA which maximized the probability of obtaining heterozygous lambs. A ssODN was designed to serve as a synonymous repair template that disrupted the PAM site for a pair of sgRNAs targeting BMPR2 exon 3 on opposite strands. We electroporated ovine zygotes six hours post-fertilization using sgRNA (100 ng/µL)/Cas9 (200 ng/µL) ribonucleoprotein (RNP) complexes alongside three ODN concentrations (100, 300, and 600 ng/µL). Zygotes were cultured for seven days, and blastocysts were lysed, PCR-amplified, and Sanger sequenced. Sequences were analyzed using TIDER (Brinkman et al., Nucl. Acids Res., 2018). TIDER is a computer model which quantifies the frequency of targeted small nucleotide changes introduced by CRISPR in combination with HDR using a donor template. A Kruskal-Wallis test compared the efficacy of HDR at 100, 300, and 600 ng/µL of ODN template. A significant difference was found between groups with 600 ng/µL giving the highest rate of HDR (P < 0.0001). Post-hoc pairwise comparisons using a Dunn's test found that sgRNA1 at 600 ng/ul ODN was significantly more efficient for HDR repair as compared with sgRNA2 (P < 0.05). The editing efficiency of the blastocysts collected using the 600 ng/µLODN and sgRNA1 treatment combination was 44.7% high (>66%), 42.6% intermediate (66-33%), and 8.5% low (< 33%) knockout rate, and 4.2% wild-type. The knockin rate was 27.7% high (>20%), 21.3% intermediate (20-5%), and 51% low to no (< 5%) HDR. These data predict that about one-half the transferred embryos subject to these conditions will have the desired heterozygous genotype [BMPR2 (+/-) sheep].

Bacterial Retrons Enable Precise Gene Editing in Human Cells.

Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve the efficiency of precise genome editing via homology-directed repair (HDR). In human cells, HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.4%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.

A repackaged CRISPR platform increases homology-directed repair for yeast engineering.

Inefficient homology-directed repair (HDR) constrains CRISPR-Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform-lowered indel nuclease system enabling accurate repair (LINEAR)-which enhanced HDR rates (to 67-100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.

Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors

BackgroundNew breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon.ResultsUsing CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively).ConclusionsIn this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.

CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus.

The ability to introduce mutations, or transgenes, of choice to precise genomic locations has revolutionized our ability to understand how genes and organisms work. In many mosquito species that are vectors of various human diseases, the advent of CRISPR genome editing tools has shed light on basic aspects of their biology that are relevant to their efficiency as disease vectors. This allows a better understanding of how current control tools work and opens up the possibility of novel genetic control approaches, such as gene drives, that deliberately introduce genetic traits into populations. Yet for the Anopheles funestus mosquito, a significant vector of malaria in sub-Saharan Africa and indeed the dominant vector species in many countries, transgenesis has yet to be achieved. We describe herein an optimized transformation system based on the germline delivery of CRISPR components that allows efficient cleavage of a previously validated genomic site and preferential repair of these cut sites via homology-directed repair (HDR), which allows the introduction of exogenous template sequence, rather than end-joining repair. The rates of transformation achieved are sufficiently high that it should be able to introduce alleles of choice to a target locus, and recover these, without the need to include additional dominant marker genes. Moreover, the high rates of HDR observed suggest that gene drives, which employ an HDR-type mechanism to ensure their proliferation in the genome, may be well suited to work in A. funestus.

A Novel Set of Cas9 Fusion Proteins to Stimulate Homologous Recombination: Cas9-HRs.

CRISPR- Cas9 has revolutionized genetic engineering. However, the inability to control double-strand break (DSB) repair has severely limited both therapeutic and academic applications. Many attempts have been made to control DSB repair choice. However, particularly in the case of larger edits, none have been able to bypass the rate-limiting step of homologous recombination (HR): long-range 5' end resection. Here, we describe a novel set of Cas9 fusions, Cas9-HRs, designed to bypass the rate-limiting step of HR repair by simultaneously coupling initial and long-range end resection. Here, we demonstrate that Cas9-HRs can increase the rate of homology directed repair (HDR) by 2- to 2.5-fold and decrease p53 mediated cellular toxicity by two- to fourfold compared to Cas9 and are functional in multiple mammalian cell lines with minimal apparent editing site bias. These properties should make Cas9-HRs an attractive option for applications demanding increased HDR rates for long inserts and/or reduced p53 pathway activation.

Homology-Directed Repair in Zebrafish: Witchcraft and Wizardry?

Introducing desired mutations into the genome of model organisms is a priority for all research focusing on protein function and disease modeling. The need to create stable mutant lines has resulted in the rapid advancement of genetic techniques over the last few decades from chemical mutagenesis and zinc finger nucleases to clustered regularly interspaced short palindromic repeats (CRISPR) and homology-directed repair (HDR). However, achieving consistently high success rates for direct mutagenesis in zebrafish remains one of the most sought-after techniques in the field. Several genes have been modified using HDR in zebrafish, but published success rates range widely, suggesting that an optimal protocol is required. In this review, we compare target genes, techniques, and protocols from 50 genes that were successfully modified in zebrafish using HDR to find the statistically best variables for efficient HDR rates.

Multiplexed Functional Assessment of Genetic Variants in CARD11

SummaryGenetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon

Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and double-stranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5′-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general.

Ex Vivo Expansion with E478 Increases the Rate of CRISPR-Mediated Homology Directed Repair of the Beta-Globin Gene in Human Hematopoietic Stem Cells By >10-Fold and Improves In Vivo Engraftment By >120-Fold

Background . Site-specific gene correction of hematopoietic stem cells (HSCs) via homology directed repair (HDR) has the potential to precisely repair defective genes and provide life-long cures for a variety of blood-based diseases. It is possible to obtain high levels of HDR during in vitro HSC culture, but these cells fail to robustly engraft in vivo, suggesting that the procedure of HDR compromises HSC function or that true HSCs are not undergoing HDR. Cells need to be actively cycling in order to undergo HDR, but conditions that allow HSC replication in vitro without compromising HSC number and function remain elusive. Thus, most HDR protocols minimize time in culture, potentially limiting HDR rates and cell yield. We recently reported that ex vivo expansion of HSCs with an aryl hydrocarbon receptor (AHR) antagonist is a clinically validated method to expand HSCs. The AHR antagonist-expanded CD34+ cell therapy, MGTA-456, results in rapid and durable recovery in patients with hematologic malignancies and inherited metabolic diseases (Wagner et al Cell Stem Cell 2016; Orchard et al AAN 2019). To apply this technology to gene-modified HSCs, we developed a novel AHR antagonist, E478, which expands NSG-engrafting cells 10-fold compared to uncultured primary human mobilized peripheral blood (mPB) CD34+cells in limit dilution studies. We previously showed that expansion with E478 results in up to 10-fold higher engraftment of lentiviral vector (LVV)-transduced cells and CRISPR/Cas9 knockout cells (Hoban et al ASGCT 2019). Here, we demonstrate that ex vivo expansion of mPB CD34+ cells with E478 results in >10-fold increase in rate of HDR and >120-fold increase in NSG engraftment of HDR+ cells compared to conventional approaches. Results . To determine whether more active cycling would lead to higher rates of HDR, we cultured cells for 1, 2, 3, and 4 days prior to electroporation with CRISPR gRNA targeting the beta-globin gene and transduction with a GFP-containing adeno-associated virus (AAV) donor template. Cell cycle analysis revealed that 33±1.8% of cells enriched for HSCs (CD34+CD90+ cells) remain quiescent after 2 days in culture, whereas 0.92±0.06% of CD34+CD90+ cells were quiescent after 3 and 4 days in culture (n=2 mPB donors). We then assessed HDR rates and HSC number after 1, 2, 3, and 4 days of additional culture. Compared to a conventional HDR protocol utilizing a 2-day pre-stimulation period followed by 1 day of culture after electroporation (herein called a 2+1 culture), we observed up to 8-fold increase in HDR with longer pre-stimulation periods, but this was accompanied with differentiation of CD34+CD90+ cells and loss of engraftment in NSG mice (79% decrease, p<0.001). We next evaluated whether E478 could increase the dose of HSCs and maintain high HDR rates. We cultured mPB CD34+ cells with E478 for a 4 day pre-stimulation, performed HDR, and continued the expansion for 4 days with E478 (herein called 4+4 culture). With the 4+4 protocol, we observed a 6-fold increase in the rate of HDR in vitro and a 134-fold increase in the number of CD34+CD90+ cells with E478 relative to 2+1 conditions with DMSO vehicle (n=2, p<0.01). Transplant of these cells into sublethally-irradiated NSG mice resulted in a 4-fold higher rate of engraftment (Figure A, p<0.01, n=8 mice), 12-fold higher rates of HDR (Figure B, p<0.001) and >120-fold increase in the number of HDR+ NSG-engrafting cells relative to 2+1 cultures (Figure C, p<0.001). Further, a 2+1 culture with E478 led to an 8-fold increase in number of HDR+ NSG-engrafting cells (p<0.001) relative to standard 2+1 approaches without a small molecule. Multi-lineage engraftment was observed in all groups. Studies using E478 with bone marrow from patients with sickle cell disease are in progress and will be presented. Conclusions. We demonstrate that ex vivo HSC expansion with E478 enables higher rates of HDR and a high dose of HDR+ HSCs, leading to >120-fold increase in the engraftment of HDR+ HSCs compared to conventional 2+1 approaches. Culture with E478 is a promising approach to realize the full potential of targeted gene correction in HSCs for a variety of genetic diseases. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. George:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs

Genome editing represents a promising strategy for the therapeutic correction of COL7A1 mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). DNA cleavage followed by homology-directed repair (HDR) using an exogenous template has previously been used to correct COL7A1 mutations. HDR rates can be modest, and the double-strand DNA breaks that initiate HDR commonly result in accompanying undesired insertions and deletions (indels). To overcome these limitations, we applied an A•T→G•C adenine base editor (ABE) to correct two different COL7A1 mutations in primary fibroblasts derived from RDEB patients. ABE enabled higher COL7A1 correction efficiencies than previously reported HDR efforts. Moreover, ABE obviated the need for a repair template, and minimal indels or editing at off-target sites was detected. Base editing restored the endogenous type VII collagen expression and function invitro. We also treated induced pluripotent stem cells (iPSCs) derived from RDEB fibroblasts with ABE. The edited iPSCs were differentiated into mesenchymal stromal cells, a cell population with therapeutic potential for RDEB. In a mouse teratoma model, the skin derived from ABE-treated iPSCs showed the proper deposition of C7 at the dermal-epidermal junction invivo. These demonstrate that base editing provides an efficient and precise genome editing method for autologous cell engineering for RDEB.

In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods

Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34+ mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12–14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ−/− Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation.

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

SummaryWe describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.

Conversion of T-Effector Cells to Immunosuppressive T-Regulatory-like Cells By CRISPR/Cas9-Mediated Integration of a FOXP3 Transgene

Regulatory T cells (Treg) are distinct among T cell subtypes, having the primary role of suppressing adaptive immune responses. The importance of these cells in immune self-tolerance is underscored by the genetically inherited syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), which is caused by an inactivating mutation in FOXP3. FOXP3 is a transcription factor that is a determinant of regulatory T cell function. Patients with IPEX syndrome suffer from the rapid and severe onset of multi-organ autoimmunity, including severe enteropathy, Type I diabetes, thyroiditis, skin inflammation and other features. In mouse models of IPEX, neonatal transplantation of wild-type Tregs is sufficient to prevent the development of disease. Less-severe Treg defects have also been implicated in the etiology of a variety of prevalent autoimmune diseases. It is possible that the pivotal role for Tregs in self-tolerance could be exploited clinically to improve therapies for autoimmunity and other diseases of tolerance. However, the use of autologous ex vivo expanded Treg as a clinical cell therapy is problematic: Tregs are present in low numbers in the peripheral blood, they expand slowly in culture ex vivo, and they may lack antigen specificities necessary for efficient suppression in specialized tissues. They may also down-regulate FOXP3 expression and lose functional activity in vivo in the setting of chronic inflammation. Additionally, autologous Tregs from patients with autoimmune disease may exhibit cell intrinsic dysfunction, while IPEX patients do not even have Tregs. To overcome these issues, we developed a gene editing approach to enforce stable expression of FOXP3 in primary human CD4+ peripheral blood T cells. CRISPR/Cas9 ribonucleoprotein and an AAV6-delivered donor template were developed to target a MND promoter-FOXP3 cDNA expression cassette (linked to a cell surface LNGFR tag by a 2A ribosome skip peptide) to the FOXP3 locus by homology directed repair (HDR). Highly efficient HDR rates were achieved across multiple donors (~34%; 5 donors in 9 experiments). For therapy of IPEX caused by FOXP3 missense mutations, integration of the functional coding sequence simultaneously abolishes endogenous FOXP3 expression. Following gene editing, expression of FOXP3 was sufficient to drive Treg-like phenotypic changes, including the up-regulation of CD25 and inhibitory receptors and down-regulation of CD127 and inflammatory cytokines. Further, consistent with the translatability of this approach into clinical manufacturing, FOXP3+ cells could be enriched to >90% purity by a simple LNGFR antibody column and expanded 20-fold within one week. Importantly, transfer of these edited Treg-like cells (edTreg) to NOD-scid-IL2Rγ-/- mice prevented xeno-graft vs. host disease (xeno-GvHD) mediated by co-transferred autologous effector T cells; xeno-GvHD protection correlated with long-term survival of the edTregs, and a marked reduction in effector T cell expansion and tissue infiltration. These data support the development of edited regulatory T cells for the treatment of IPEX and other autoimmune disease. DisclosuresScharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership.

CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments.

ABSTRACTTargeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.This article has an associated First Person interview with the first author of the paper.

Improving CRISPR-Cas9’s Rate of Homology-Directed Repair

Genetic Engineering & Biotechnology NewsVol. 38, No. 14 Drug Discovery TutorialImproving CRISPR-Cas9’s Rate of Homology-Directed RepairIntegrated DNA Technologies Suggests Strategies to Boost Efficiency, Avoid Blunt-End Incorporation, and Reduce ToxicityBrian WangBrian WangBrian Wang, Ph.D. (E-mail Address: bwang@idtdna.com), is a genomic tools market development manager at Integrated DNA Technologies. Website: www.idtdna.com.Search for more papers by this authorPublished Online:27 Jul 2018https://doi.org/10.1089/gen.38.14.06AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 38Issue 14Aug 2018 InformationCopyright © GEN PublishingTo cite this article:Brian Wang.Improving CRISPR-Cas9’s Rate of Homology-Directed Repair.Genetic Engineering & Biotechnology News.Aug 2018.12-13.http://doi.org/10.1089/gen.38.14.06Published in Volume: 38 Issue 14: July 27, 2018PDF download

\u2018Cold shock\u2019 increases the frequency of homology directed repair gene editing in induced pluripotent stem cells

Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20–40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1–20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by ‘cold shocking’ cells at 32 °C for 24–48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or ‘perfect HDR’, as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated (‘partial HDR’). We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing ‘perfect HDR’ compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines.

Therapeutic Crispr/Cas9 Genome Editing for Treating Sickle Cell Disease

IntroductionSickle cell disease (SCD) is one of the most common monogenic disorders, affecting millions worldwide. SCD is caused by a point mutation in the β-globin gene (HBB). A single nucleotide substitution from A to T in the codon for the sixth amino acid in the β-globin protein converts a glutamic acid to a valine that leads to the production of sickle hemoglobin (HbS), which impairs the function of the red blood cells (RBCs). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but it is feasible for only a small subpopulation (<15%) of patients and may be associated with a high risk. Here, we show that targeted genome editing can potentially provide a permanent cure for SCD by correcting the sickle mutation in clinically relevant hematopoietic stem and progenitor cells (HSPCs) for autologous transplantation.MethodsFor proof-of-concept, we designed CRISPR/Cas9 systems and donor templates to introduce the sickle mutation into wild-type (WT) HBB of mobilized peripheral blood CD34+ cells. To assess genome-editing outcomes mediated by CRISPR/Cas9 systems, we developed a novel digital droplet PCR (ddPCR) assay that can quantify the rates of non-homologous end joining (NHEJ) and homology directed repair (HDR) events simultaneously following the generation of DNA double strand breaks. The assay enables rapid and accurate quantification of gene modifications in HSPCs by CRISPR/Cas9 genome-editing. Specifically, Streptococcus pyogenes (Spy) Cas9 proteins, guide RNAs (gRNA), and single-stranded DNA (ssDNA) donor templates were delivered into CD34+ cells by nucleofection with optimized conditions. Different gRNAs targeting HBB near the SCD mutation site were tested, and the optimal gRNA was chosen based on high on-target activity and proximity to the mutation site. The optimal DNA donor design and concentration were determined based on the frequency of HDR events and viability/growth rate of edited cells. Treated samples and untreated controls were assayed as both single cell clones and in bulk culture. In 2-phase liquid culture, genome editing frequencies at both DNA and mRNA levels were quantified by ddPCR to confirm persistence of edited cells in the heterozygous population over time. The expression of globins and other erythroid markers were monitored using flow cytometry and real time PCR to determine if genome editing had any effect on the kinetics of erythropoiesis. Colony formation assays were used to determine the number and type of colonies following induction of differentiation. Colony ddPCR was performed to determine the genotype of edited cells. Wright/Giemsa stain was used to confirm terminal maturation of erythrocytes into enucleated RBC. Native polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC) were used to confirm translation of edited β-globin protein and formation of HbS.Results and DiscussionWe found that the efficiency of site-specific gene correction could be substantially improved by optimizing the CRISPR/Cas9 systems for genome editing. For example, with optimization, we achieved ~30% HDR rates in CD34+ cells with >80% cell viability. The HDR-modified alleles persisted in the population over the course of differentiation, and the edited CD34+ cells retained differentiation potential. Genotyping of individual erythroid colonies confirmed that up to 35% of colonies are either homozygous or heterozygous for HDR alleles. Following differentiation, treated cells express modified HBB mRNA and HbS. In addition, the off-target activity of the HBB-specific gRNAs was determined using both bioinformatics tools and unbiased genome-wide mapping techniques. Ongoing work includes the validation of gene correction in SCD patient derived HSPCs, characterization of modified cells in vitro and in vivo to assess the therapeutic potential, and analysis of long-term genotoxicity.ConclusionsBased on the proof-of-concept study, we demonstrate that using the optimized CRISPR/Cas9 system and donor template, an HDR rate of ~30% can be achieved in CD34+ cells. The gene corrected cells have the potential to differentiate into erythroid cells that permanently produce WT β-globin. Our findings provide promising evidence for clinical translation of the HSPCs genome correction strategy in treating SCD patients, as well as correcting gene defects underlying other inherited single-gene disorders. DisclosuresNo relevant conflicts of interest to declare.

Abstract 71: Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair

Abstract The comprehensive and coordinated efforts of The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) identified the somatic genomic alterations occurring in a broad panel of human cancers. The challenge ahead is to understand the functions of these somatically altered genes.Genome editing tools like the CRISPR/Cas9 system represent a transformative technology that allows the precise engineering of cells harboring specific (point) mutations found in specific tumor subtypes, enabling the investigation of the role of a candidate cancer gene in a stringently defined genetic context. Once recruited to its DNA target sequence in cells, Cas9 induces a DNA double strand break (DSB), which is either repaired by the non-homologous end-joining pathway (NHEJ), or by homology-directed repair (HDR). The HDR pathway allows for a precise site-specific knock-in and thus functional analysis of cancer-associated sequence variants. However, HDR activity is restricted to the late S and G2 phases of the cell cycle, while the NHEJ pathway dominates DNA repair throughout the cell cycle. Thus, the error-prone NHEJ pathway out-competes the HDR pathway, which reduces the likelihood for precise insertions, deletions or base substitutions at the DSB. Here, we devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1 resulting in a cell cycle tailored expression with low levels in G1, but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the relative rate of HDR by up to 87% at a single-copy reporter locus, and up to 42% at the endogenous MALAT1 locus compared to wild-type Cas9. In summary, our regulatory concept takes advantage of cell-autonomous pathways to enable fluctuation of Cas9 protein levels coupled to cell cycle dynamics. This resulted in higher site-specific integration events without sophisticated manipulation of cells by small molecules or additional gene targeting reagents. Future methodological developments may enable high-resolution expression of Cas9 and alternative genome engineering proteins like Cpf1 by combining cell cycle phase-specific transcriptional and post-translational regulatory elements. This might enhance HDR efficiencies even further, and will help to make sense of cancer genomic data. Citation Format: Tony Gutschner, Monika Hämmerle, Giannicola Genovese, Giulio F. Draetta, Lynda Chin. Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 71.