Donor DNA Template Research Articles

123. Gene Editing as a Therapeutic Approach to Treat IPEX Syndrome

Site-specific gene editing combined with autologous stem cell transplantation holds tremendous potential to treat both inherited and acquired diseases. However, the translational applications of gene editing are limited by the challenge of achieving sufficient levels of gene targeting in human primary cells. Recent advances in gene editing, including use of the CRISPR-Cas9 system, nuclease-resistant chemically modified small guide RNAs (sgRNAs), and serotype-optimized AAV delivery of donor repair DNA templates, are enabling clinically relevant levels of gene editing in human primary cells. Here, we build upon these advances to develop a gene editing strategy to treat the immune-dysregulation polyendocrinopathy-enteropathy-X-linked (IPEX) syndrome, a severe primary immune deficiency manifesting with life-threatening multi-organ autoimmunity in children. IPEX is caused by mutations in the forkhead box protein 3 gene (FOXP3), resulting in dysfunction of T regulatory cells (Tregs) and T effector cell lymphoproliferation. Due to the presence of disease causative mutations throughout the entire FOXP3 gene and the complex nature of FOXP3 regulatory elements, we designed a strategy to functionally correct the gene in IPEX patient cells using on-target, homology directed repair (HDR)-mediated insertion of the FOXP3 coding sequence into the gene locus. By targeting the FOXP3 gene using a CRISPR system comprised of Cas9 mRNA and chemically modified sgRNAs, we were able to attain high targeting frequencies, reaching 70-80% in human primary CD4+ T cells. We use this CRISPR system in combination with a repair donor DNA template delivered with AAV to demonstrate FOXP3 gene correction in human CD34+ hematopoietic stem and progenitor cells (HSPCs). In addition to gene correction of FOXP3, we use a similar strategy in parallel to knockout FOXP3 in wild-type human CD34+ HSPCs. We use the knockout HSPCs to generate a humanized mouse model of FOXP3 dysfunction, which enables us to study the pathophysiology of IPEX and to ultimately test the efficacy of gene-corrected Treg cell-based therapies. These results will help demonstrate the feasibility of FOXP3 gene editing, which we propose for the translational application of autologous transplant of Tregs and HSPCs as a therapy for IPEX syndrome.

42. Correction of SCID-X1 by Targeted Genome Editing of Hematopoietic Stem/Progenitor Cells (HSPC) in the Mouse Model

Targeted genome editing by engineered nucleases has brought the goal of gene correction within the reach of gene therapy. A candidate disease for HSPC gene correction is SCID-X1, because gene therapy trials with integrating vectors showed robust clinical efficacy even from few corrected cells but also the occurrence of leukemias due to insertional mutagenesis and unregulated transgene expression. To model SCID-X1 gene correction in preclinical studies, we developed a mouse model carrying the IL2RG human gene harboring a common disease-causing mutation in place of the murine Il2rg, allowing to use of the same reagents developed for gene correction of human cells. These mice have impaired lymphoid development which phenocopies that reported for Il2rg-/- mice. To assess the minimal level of corrected HSPC required to achieve immune reconstitution we performed competitive transplants with wild-type (WT) and Il2rg-/- HSPC and found that 1% of WT cells are sufficient to reconstitute in part the T and B cell compartments. We then tested gene correction of the murine Lin- HSPC by the delivery of donor DNA template by IDLVs followed by transfection of ZFN mRNAs. This protocol yielded high on-target nuclease activity (40%) and a mean of 6% transgene integration by HDR but also high cytotoxicity (65% cell loss) under the conditions we used. The surviving cells remained capable of expansion in culture and maintained their clonogenic potential. Importantly, upon transplant into lethally irradiated mice, only the gene corrected cells were able to generate lymphoid lineages (B and T cells), showing a clear selective advantage over the un-corrected SCID cells. These data indicate functional correction of the defective IL2RG gene by targeted editing. Furthermore, upon challenging the mice with a murine pathogen we observed viral-specific γIFN production by CD8+ gene corrected cells, proving their in vivo functionality. Yet, measuring the percentage of edited cells (either by NHEJ or HDR) within the HSC compartment long-term, we found that it was nearly undetectable. Despite the lack of HSC marking, gene corrected lymphoid cells persisted in the mice up to 7 months post transplantation within all the hematopoietic organs, indicating successful editing of at least 1% progenitors able to sustain long-term lymphopoiesis and partially correct the disease phenotype. We then developed a new protocol exploiting CRISPR/Cas9 technology that enabled to achieve substantial levels of targeted DNA repair by NHEJ (up to 70%) and HDR (up to 25%) with minimal cytotoxicity and provided stable engraftment of the edited cells in transplanted mice. By this strategy we are now assessing the impact of HSC vs. progenitor targeted editing and conditioning regimen for the extent and stability of disease correction. These studies will help establish the key factors underlying safe and effective rescue of the disease by HSPC gene editing and assist in the design of the protocol for its first clinical testing.

Programmable Site-Specific Nucleases for Targeted Genome Engineering in Higher Eukaryotes.

Recent advances in the targeted genome engineering enable molecular biologists to generate sequence specific modifications with greater efficiency and higher specificity in complex eukaryotic genomes. Programmable site-specific DNA cleavage reagents and cellular DNA repair mechanisms have made this possible. These reagents have become powerful tools for delivering a site-specific genomic double-strand break (DSB) at the desired chromosomal locus, which produces sequence alterations through error-prone non-homologous end joining (NHEJ) resulting in gene inactivations/knockouts. Alternatively, the DSB can be repaired through homology-directed repair (HDR) using a donor DNA template, which leads to the introduction of desired sequence modifications at the predetermined site. Here, we summarize the role of three classes of nucleases; zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system in achieving targeted genome modifications. Further, we discuss the progress towards the applications of programmable site-specific nucleases (SSNs) in treating human diseases and other biological applications in economically important higher eukaryotic organisms such as plants and livestock. J. Cell. Physiol. 231: 2380-2392, 2016. © 2016 Wiley Periodicals, Inc.

A Double Strand Break during Telophase Is Repaired with Homologous Recombination Despite the Absence of an Available Sister Chromatid

Background:Chromosomal breakage results from a DNA double strand break (DSB), and is repaired to maintain and restore genetic integrity, principally through two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by nucleolytic resection of a DSB in the presence of cyclin-dependent kinase 1 (Cdk1) activity. DSB repair through HR is dependent on Rad52, and can be error-free when a sister chromatid is used as a template for repair. However, HR is mutagenic when any other template is used for repair. Loss of nucleotides adjacent to the DSB is a feature of repair through NHEJ. There is co-relation between Cdk1 activity and the presence of a sister chromatid. The research question was, in addition to Cdk1 activity is the presence of an intact sister chromatid a requirement to initiate DSB repair with the HR pathway.Methods:Cdk1 activity peaks during mitosis in the presence of an intact sister chromatid. To study DSB resection and repair in cells arrested in either mitotic metaphase or telophase when Cdk1-Clb2 was active, conditional alleles were constructed in a eukaryotic haploid budding yeast model of HR. The model permitted simultaneous induction of a single site-specific DSB in cells that were synchronised to a phase of the cell division cycle. Physical monitoring of the kinetics of DSB formation, nucleolytic resection of adjacent DNA, and DSB repair, was achieved by probing Southern membranes after restriction enzyme digestion of extracted genomic DNA from time courses.Results:Sister chromatids were segregated during telophase arrest induced by either Cdc14 or Cdc15 depletion. Metaphase arrest was achieved with Cdc20 depletion, either directly, or indirectly by activation of the spindle assembly checkpoint by inhibition of microtubule polymerisation. Sister chromatids were unsegregated and physically attached through cohesin during metaphase.The absence of an intact sister chromatid did not prevent DSB repair with the HR pathway during telophase. Nucleolytic resection was observed in the presence or absence of an intra-chromosomal homologous but non-identical DNA repair template. The DSB cut site did not become resistant to cycles of re-cleavage through loss of adjacent nucleotides. DSB repair by HR was dependent on Rad52. The kinetics of nucleolytic resection adjacent to the DSB, and repair by HR, were similar during telophase and metaphase.Conclusions:This is the first study to report the observation that the availability of the sister chromatid is not a requirement to promote DSB repair with the HR pathway during telophase. Initiation of HR occurs despite segregated sister chromatids, even in the absence of a non-identical homologous DNA donor template, with inherently mutagenic repair by HR. This unexpected discovery has important clinical implications to the pathogenesis of chromosomal translocations and oncogenesis, and tumour progression with repair of treatment-induced DSBs. DisclosuresNo relevant conflicts of interest to declare.

Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.

Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by AAV serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome editing technologies in HSPCs.

478. Highly Efficient Targeted Gene Addition in CD34+ Hematopoietic Stem/Progenitor Cells by Combining ZFN mRNA and AAV6 Donor Delivery

Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene correction/addition, respectively. Hematopoietic stem/progenitor cells (HSPC) are an important clinical target for gene therapy. These cells can be readily harvested from the subject, engineered using viral vectors, and re-introduced back into the patient where they give rise to all of the mature cells of the blood and immune system. Thus the ability to edit the genome of HSPCs could provide a corrective therapy for a number of monogenic diseases. However, achieving high levels of editing by HDR may be significantly more challenging than gene knockout by NHEJ. Here we report that the combination of delivering the ZFNs as mRNA and corrective donor DNA via a rAAV6 vector can induce highly efficient targeted gene addition in CD34+ HSPCs.Using CCR5-specific ZFNs and donor DNA templates as a model system we show that targeted addition of a novel restriction fragment length polymorphism (RFLP) was consistently achieved at >15%, while insertion of an eGFP expression cassette was achieved at >10% of the CCR5 loci in mobilized CD34+ cells. Furthermore, targeting the AAVS1 “safe harbor” locus with a pair of AAVS1-specific ZFNs (mRNA) and a RFLP donor (rAAV6) resulted in >30% of AAVS1 alleles specifically being edited to include the RFLP, suggesting this genome editing approach to achieve highly efficient targeted gene addition in CD34+ cells can be applied to different genetic loci. Edited HSPC undergo normal hematopoiesis in both in vitro differentiation assays and during engraftment of NSG mice. Using fetal liver derived cells, editing of primitive and more committed CD34+ HSPC subsets was observed at similar levels in HSPC CFU cultures, and by analysis of sorted populations of treated HSPC, including LT-HSC, and through the secondary transplantation of NSG mice.The use of ZFNs in combination with rAAV6 delivery of the donor DNA provides a significant advance in genome editing that has great potential for targeted correction of human disease-causing mutations and permanent transgene addition in human CD34+ HSPC.

481. Targeted Genome Editing in Mouse Hematopoietic Stem/Progenitor Cells (HSPC) To Model Gene Correction of SCID-X1

Targeted genome editing by artificial nucleases has brought the goal of gene correction within the reach of gene therapy. A candidate disease for HSPC gene correction is SCID-X1, because gene therapy with early generation integrating vectors showed robust clinical efficacy even from few corrected cells but also the occurrence of adverse events due to insertional mutagenesis and unregulated transgene expression. We recently reported a strategy that enabled targeted integration of a corrective cDNA into the IL2RG gene in 6% of human HSPC with high specificity. Gene corrected HSPC generated polyclonal lymphoid cells that express the IL2RG protein and have a selective growth advantage over those carrying disruptive IL2RG mutations (Genovese, Nature, 2014). Here, to model SCID-X1 disease correction, we developed a mouse model carrying the IL2RG human gene including a common disease-causing mutation in place of the murine Il2rg gene, allowing to use the same reagents utilized for gene correction of human cells. These mice have impairment in lymphoid development which phenocopies that reported for Il2rg-/- mice. To assess the minimal level of corrected HSPC required to achieve immune reconstitution we first performed competitive transplants with wild-type (WT) and Il2rg-/- HSPC and found that 1% of WT cells are sufficient to reconstitute at least in part the T and B cell compartments. We then developed a protocol to obtain gene correction in murine Lin- HSPC based on the delivery of donor DNA template by IDLVs followed by transfection of ZFN mRNAs. This protocol was associated with high on-target nuclease activity (40%) and a mean of 6% transgene integration by HDR, but also with high levels of acute cytotoxicity (65% cell loss). The surviving cells remained capable of expansion in culture and preserved their clonogenic potential. Importantly, upon transplant into lethally irradiated mice, only the gene corrected cells were able to generate lymphoid lineages (B and T cells), showing a clear selective advantage over un-corrected cells. These data indicate functional correction of the IL2RG gene by our strategy. Yet, measuring percentage of correction within myeloid cells at long-term we found that it was almost undetectable. Despite the lack of HSC marking, gene corrected lymphoid cells stably persisted in the mice up to 7 months post transplantation within all the hematopoietic organs. Furthermore, upon challenging the transplanted mice with a murine pathogen (LCMV Arm.) we observed viral-specific γIFN production by CD8+ gene corrected cells at a similar extent as for WT mice, proving in vivo the functionality of corrected T cells. These results suggest that our protocol achieves biologically relevant levels of gene correction in progenitors that sustain long-term lymphopoiesis but is limited in multipotent HSC. Ongoing studies aim to improve murine HSC gene targeting and to compare safety and efficacy of gene correction vs gene replacement in our disease model.

PM-11 * DEVELOPMENT OF A NOVEL GENE TARGETING AND CLONE SCREENING PLATFORM TO ENGINEER COMMON PEDIATRIC GLIOMA MUTATIONS INTO MODEL CELL LINES

The utility of CRISPR/Cas9 gene targeting tools to alter endogenous genomic loci has revolutionized biomedical research across numerous disease focus areas, with especially large gains made in the development of disease models. However, the introduction of specific mutations using donor DNA templates can be associated with low yields of single cell clones harboring homozygous versus heterozygous mutations. Conventional clone screening methods can be a labor intensive and expensive process, and thus better approaches are needed to identify cells with the desired genotype(s). In parallel, there is a dearth of human tumor cell model systems for pediatric cancers. To address these needs, we recently designed and validated a platform which utilizes a novel, inducible Cas9 expression system coupled with a high-resolution DNA melt (HRM) analysis protocol to rapidly identify mutant clones. We applied our platform to model several common DIPG mutations, including the Lys27Met missense mutation within histone variant H3.3 (H3F3A), and truncating mutations within protein phosphatase 1D (PPM1D) exon 6. Using our HRM protocol, both heterozygous point mutations in H3F3A, and indels in PPM1D were detectable and grouped distinctly from wild-type samples. These findings were validated using conventional Sanger sequencing and topo cloning, which confirmed that this approach could differentiate between mutant and wild-type clones. Multiple clones were generated in under four weeks using this process-flow. These findings highlight the utility and power of our unique approach to rapidly create engineered mutant cell lines. Our platform likely will become an invaluable tool for the development of better pediatric glioma cell line models in the future.

Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation.

Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa β-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470-1479.

Adenoviral vector DNA for accurate genome editing with engineered nucleases.

Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.

Targeted genome correction by a single adenoviral vector simultaneously carrying an inducible zinc finger nuclease and a donor template

Zinc finger nuclease (ZFN) technology, which can be used to induce targeted genome correction in the presence of a DNA donor template, is becoming an attractive strategy for treating monogenic diseases. This strategy requires efficient delivery of ZFN and donor template into cells, ideally, in a single viral vector to achieve efficient genome editing and to avoid unwanted mutagenesis. In this study, we successfully produced a single adenoviral (Ad) vector with high titer that carried a ZFN expression cassette and a donor template simultaneously. We then demonstrated that this single Ad system could mediate efficient site-specific genome correction in vitro and ex vivo. The gene correction efficiency of the single Ad was significantly higher than that of the double Ad system. This novel vector will be a promising ZFN and donor delivery system for treatment of monogenic diseases.

Turning selfish proteins into programmable genome editors (733.2)

Though no suitable methods for genome editing yet exist, the most promising approaches utilize targeted nucleases and take advantage of a cell’s natural repair processes that either disrupts the cleaved sequence or repairs it using a donor DNA template. LAGLIDADG Homing Endonucleases (LHE’s) are naturally occurring selfish proteins with two key features ideal for genome editing: recognition of a long DNA target site (~22 bp) and double‐stranded cleaving activity. Originally thought to be impossible to engineer due to their size and complexity, recent approaches based on experimentally determined structural models to direct the bioengineering process have proven successful. The aim of my research project was to solve the three dimensional structures of various LHE’s using x‐ray crystallography. Using E. coli‐expressed proteins, I solved the structures of two LHE’s (I‐GpeMI and I‐CpaMI) bound to their target DNA. With these structures we are able to identify DNA‐interacting amino acids and focus our engineering efforts of the enzyme scaffolds towards new DNA target sites.Grant Funding Source: Murdock Trust

Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila.

We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.

Targeted transgene insertion into the AAVS1 locus driven by baculoviral vector‐mediated zinc finger nuclease expression in human‐induced pluripotent stem cells

The AAVS1 locus is viewed as a 'safe harbor' for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy.

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis.

Genome engineering in human pluripotent stem cells (hPSCs) holds great promise for biomedical research and regenerative medicine. Recently, an RNA-guided, DNA-cleaving interference pathway from bacteria [the type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) pathway] has been adapted for use in eukaryotic cells, greatly facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus pyogenes and Streptococcus thermophilus), each with their own distinct targeting requirements and limitations, have been developed for genome editing thus far. Furthermore, limited information exists about homology-directed repair (HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis, we demonstrate efficient targeting of an endogenous gene in three hPSC lines using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) recognizes a 5'-NNNNGATT-3' protospacer adjacent motif (PAM) different from those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9 and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a single-guide RNA (sgRNA) to direct its activity. Because of its distinct protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases the sequence contexts amenable to RNA-directed genome editing.

Generation of an HIV Resistant T-cell Line by Targeted “Stacking” of Restriction Factors

Restriction factors constitute a newly appreciated line of innate immune defense, blocking viral replication inside of infected cells. In contrast to these antiviral proteins, some cellular proteins, such as the CD4, CCR5, and CXCR4 cell surface receptors, facilitate HIV replication. We have used zinc finger nucleases (ZFNs) to insert a cocktail of anti-HIV restriction factors into the CCR5 locus in a T-cell reporter line, knocking out the CCR5 gene in the process. Mirroring the logic of highly active antiretroviral therapy, this strategy provides multiple parallel blocks to infection, dramatically limiting pathways for viral escape, without relying on random integration of transgenes into the genome. Because of the combination of blocks that this strategy creates, our modified T-cell lines are robustly resistant to both CCR5-tropic (R5-tropic) and CXCR4-tropic (X4-tropic) HIV-1. While zinc finger nuclease-mediated CCR5 disruption alone, which mimics the strategy being used in clinical trials, confers 16-fold protection against R5-tropic HIV, it has no effect against X4-tropic virus. Rhesus TRIM5α, chimeric human-rhesus TRIM5α, APOBEC3G D128K, or Rev M10 alone targeted to CCR5 confers significantly improved resistance to infection by both variants compared with CCR5 disruption alone. The combination of three factors targeted to CCR5 blocks infection at multiple stages, providing virtually complete protection against infection by R5-tropic and X4-tropic HIV.

Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects.

Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.

Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells.

The exchange of genetic information between donor and acceptor DNA molecules by homologous recombination (HR) depends on the cleavage of phosphodiester bonds. Although double-stranded and single-stranded DNA breaks (SSBs) have both been invoked as triggers of HR, until very recently the focus has been primarily on the former type of DNA lesions mainly due to the paucity of SSB-based recombination models. Here, to investigate the role of nicked DNA molecules as HR-initiating substrates in human somatic cells, we devised a homology-directed gene targeting system based on exogenous donor and chromosomal target DNA containing recognition sequences for the adeno-associated virus sequence- and strand-specific endonucleases Rep78 and Rep68. We found that HR is greatly fostered if a SSB is not only introduced in the chromosomal acceptor but also in the donor DNA template. Our data are consistent with HR models postulating the occurrence of SSBs or single-stranded gaps in both donor and acceptor molecules during the genetic exchange process. These findings can guide the development of improved HR-based genome editing strategies in which sequence- and strand-specific endonucleolytic cleavage of the chromosomal target site is combined with that of the targeting vector.

Nonspaced inverted DNA repeats are preferential targets for homology-directed gene repair in mammalian cells

DNA repeats constitute potential sites for the nucleation of secondary structures such as hairpins and cruciforms. Studies performed mostly in bacteria and yeast showed that these noncanonical DNA structures are breakage-prone, making them candidate targets for cellular DNA repair pathways. Possible culprits for fragility at repetitive DNA sequences include replication and transcription as well as the action of structure–specific nucleases. Despite their patent biological relevance, the parameters governing DNA repeat-associated chromosomal transactions remain ill-defined. Here, we established an episomal recombination system based on donor and acceptor complementary DNA templates to investigate the role of direct and inverted DNA repeats in homologous recombination (HR) in mammalian cells. This system allowed us also to ascertain in a stringent manner the impact of repetitive sequence replication on homology-directed gene repair. We found that nonspaced DNA repeats can, per se, engage the HR pathway of the cell and that this process is primarily dependent on their spacing and relative arrangement (i.e. parallel or antiparallel) rather than on their sequence. Indeed, our data demonstrate that contrary to direct and spaced inverted repeats, nonspaced inverted repeats are intrinsically recombinogenic motifs in mammalian cells lending experimental support to their role in genome dynamics in higher eukaryotes.