Multiplex Gene Editing Research Articles

Generation of Memory Stem T Cells Specific for Tumor Antigens and Resistant to Inhibitory Signals By Genome Editing

Tumor-infiltrating lymphocytes (TILs) may express different inhibitory receptors (IRs) on the cell surface that are exploited by cancer cells to evade the immune attack. Thus, exhausted T cells display an unresponsive functional profile and fail to kill tumor cells. TIM-3 and LAG-3 represent highly expressed inhibitory receptors in exhausted T cells infiltrating solid tumors. We recently showed that a large fraction of bone marrow infiltrating CD8+ cells co-express multiple inhibitory receptors, including TIM-3, in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplantation. These observations advocate for strategies to generate innovative tumor-specific T cell products resistant to inhibitory signals and able to persist long-term in treated patients. To reach these objectives we developed a strategy to simultaneously redirect T cell specificity by TCR gene transfer and permanently disrupt IRs by CRISPR/Cas9 system in long-living memory stem T cells (TSCM) for adoptive cell therapy.Primary T cells were activated with CD3/CD28-conjugated beads and cultured with low doses of IL7 and IL15, to preserve their TSCM phenotype, and electroporated with Cas9/gRNA ribonucleoproteins (RNPs) targeting a coding sequence of TIM-3,LAG3, 2B4 and of the TCR α and β chain constant region (TRAC and TRBC1/2) genes. Each gene was edited alone or in combination in a multiplex approach. The efficiencies of NHEJ-mediated inactivation of each gene were assessed by cytofluorimetric analysis and confirmed at molecular level by surveyor assay or ddPCR.In order to avoid mispairing between the endogenous TCR and the tumor-specific TCR, we simultaneously inactivated the constant regions of α and β chain of the endogenous TCR genes. Efficiency of TRAC and/or TRBC1/2 gene disruption was 98%, when measured as % of CD3neg cells. TCR knocked-out cells could be efficiently (70-85%) transduced with a lentiviral vector encoding for TCR specific for an HLA-A2 restricted peptide from NY-ESO1.We then focused on IR gene inactivation. Single gene editing produced up to 98%, 93% and 63% genetic knock-out of the TIM-3 , the LAG-3 and the 2B4 genes, respectively. We finally combined one IR inactivation with TCR gene editing in a single protocol. We obtained an average of 68% and 69% of TIM3neg and LAG3neg NY-ESO1-TCR redirected T cells, respectively, and more than 90% of edited cells showed a TSCM phenotype.When tested in functional assays, TCR-IR-edited TSCM cells proved highly effective and specific in killing HLA-A2+ NY-ESO1+ multiple myeloma cells. Of notice, TIM-3neg and LAG-3neg cells proved more effective in producing IFNg, TNFa and CD107a, once challenged with tumor cells.In conclusion, by combining the versatility of multiplex gene editing by CRISPR/Cas9 with culture conditions designed to engineer TSCM cells, we can generate innovative tumor-specific cellular products redirected against tumor antigens and resistant to inhibitory signals. Ongoing in vivo evaluation of tumor-specific IR disrupted memory stem T cells will be reported and discussed. DisclosuresBonini:Intellia Therapeutics: Research Funding.

Clonal expansion and myeloid leukemia progression modeled by multiplex gene editing of murine hematopoietic progenitor cells

Recent advances in next-generation sequencing have identified novel mutations and revealed complex genetic architectures in human hematological malignancies. Moving forward, new methods to quickly generate animal models that recapitulate the complex genetics of human hematological disorders are needed to transform the genetic information to new therapies. Here, we used a ribonucleoprotein-based CRISPR/Cas9 system to model human clonal hematopoiesis of indeterminate potential and acute myeloid leukemia (AML). We edited multiple genes recurrently mutated in hematological disorders, including those encoding epigenetic regulators, transcriptional regulators, and signaling components in murine hematopoietic stem/progenitor cells. Tracking the clonal dynamics by sequencing the indels induced by CRISPR/Cas9 revealed clonal expansion in some recipient mice that progressed to AML initiated by leukemia-initiating cells. Our results establish that the CRISPR/Cas9-mediated multiplex mutagenesis can be used to engineer a variety of murine models of hematological malignancies with complex genetic architectures seen in human disease.

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

CRISPR/Cas9 in plants: at play in the genome and at work for crop improvement.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system uses single-guide RNAs for genome editing, making it a simple, robust, powerful tool for targeted gene mutagenesis, knockout and knock-in/replacement, as well as transcriptional regulation. Here, we review the working principles, components and potential modifications of CRISPR/Cas9 for efficient single and multiplex gene editing in plants. We also describe recent work that has used CRISPR/Cas9 to improve economically important traits in crop plants. Although the apparent ease of CRISPR/Cas9-mediated editing may make it appear as though scientists are merely playing with plant genomes, the combined power of CRISPR/Cas9 has enabled vital research to be completed in the battle toward optimization and adaptation of crop species, permitting crucial advances to be achieved in crop improvement.

Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat.

The CRISPR-Cas9-based multiplexed gene editing (MGE) provides a powerful method to modify multiple genomic regions simultaneously controlling different agronomic traits in crops. We applied the MGE construct built by combining the tandemly arrayed tRNA–gRNA units to generate heritable mutations in the TaGW2, TaLpx-1, and TaMLO genes of hexaploid wheat. The knockout mutations generated by this construct in all three homoeologous copies of one of the target genes, TaGW2, resulted in a substantial increase in seed size and thousand grain weight. We showed that the non-modified gRNA targets in the early generation plants can be edited by CRISPR-Cas9 in the following generations. Our results demonstrate that transgenerational gene editing activity can serve as the source of novel variation in the progeny of CRISPR-Cas9-expressing plants and suggest that the Cas9-inducible trait transfer for crop improvement can be achieved by crossing the plants expressing the gene editing constructs with the lines of interest.

Single and Multiplexed Gene Editing in Ustilago maydis Using CRISPR-Cas9.

The smut fungus Ustilago maydis is an established model organism for elucidating how biotrophic pathogens colonize plants and how gene families contribute to virulence. Here we describe a step by step protocol for the generation of CRISPR plasmids for single and multiplexed gene editing in U. maydis. Furthermore, we describe the necessary steps required for generating edited clonal populations, losing the Cas9 containing plasmid, and for selecting the desired clones.

Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins.

SummaryPlant‐produced glycoproteins contain N‐linked glycans with plant‐specific residues of β(1,2)‐xylose and core α(1,3)‐fucose, which do not exist in mammalian‐derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant‐expressed proteins. We knocked out the β(1,2)‐xylosyltranferase (XylT) and the α(1,3)‐fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked‐out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N‐linked glycans lacking β(1,2)‐xylose and/or α(1,3)‐fucose. The knocked‐out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild‐type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco‐engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.

Erratum: Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.

Nat. Biotechnol. 35, 31–34 (2017); published online 5 December 2016; corrected after print 12 January 2017 In the version of this article initially published, in Fig. 2j, the percentage for the targets Mecp2, Nlgn3, and Drd1 should be 15.2%, not 16.9%; the same error appeared in the main text, next to last paragraph, “Our results show that ∼17%.

Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co-expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA-gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen-activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR-edited mutants. The true knock-out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR-induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45-86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene-free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.

Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA.

Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300core fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter.

P7003 Heritable multiplex gene editing via CRISPR/Cas9 exhibits no detectable genome-wide off-target effects in sheep

P7003 Heritable multiplex gene editing via CRISPR/Cas9 exhibits no detectable genome-wide off-target effects in sheep

Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep.

The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here, we successfully achieved precise gene targeting in sheep by co-injecting one-cell-stage embryos with Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2). We carefully examined the sgRNAs:Cas9-mediated targeting effects in injected embryos, somatic tissues, as well as gonads via cloning and sequencing. The targeting efficiencies in these three genes were within the range of 27–33% in generated lambs, and that of simultaneously targeting the three genes was 5.6%, which demonstrated that micro-injection of zygotes is an efficient approach for generating gene-modified sheep. Interestingly, we observed that disruption of the MSTN gene resulted in the desired muscle hypertrophy that is characterized by enlarged myofibers, thereby providing the first detailed evidence supporting that gene modifications had occurred at both the genetic and morphological levels. In addition, prescreening for the off-target effect of sgRNAs was performed on fibroblasts before microinjection, to ensure that no detectable off-target mutations from founder animals existed. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by simultaneously targeting multiple genes that are responsible for economically significant traits.

High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize.

BackgroundCRISPR/Cas9 genome editing strategy has been applied to a variety of species and the tRNA-processing system has been used to compact multiple gRNAs into one synthetic gene for manipulating multiple genes in rice.ResultsWe optimized and introduced the multiplex gene editing strategy based on the tRNA-processing system into maize. Maize glycine-tRNA was selected to design multiple tRNA-gRNA units for the simultaneous production of numerous gRNAs under the control of one maize U6 promoter. We designed three gRNAs for simplex editing and three multiple tRNA-gRNA units for multiplex editing. The results indicate that this system not only increased the number of targeted sites but also enhanced mutagenesis efficiency in maize. Additionally, we propose an advanced sequence selection of gRNA spacers for relatively more efficient and accurate chromosomal fragment deletion, which is important for complete abolishment of gene function especially long non-coding RNAs (lncRNAs). Our results also indicated that up to four tRNA-gRNA units in one expression cassette design can still work in maize.ConclusionsThe examples reported here demonstrate the utility of the tRNA-processing system-based strategy as an efficient multiplex genome editing tool to enhance maize genetic research and breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0289-2) contains supplementary material, which is available to authorized users.

Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system

The parthenotes represent ideal models mimicking the embryonic development and characterizing the function of maternal genomes as well as an alternative source of pluripotent cell lines. Besides, parthenogenetically activated (PA) embryos serve as a rapid assay system to maximize the efficiency of generating genetically modified pig CRISPR/Cas9 system, an efficient and multiplex gene editing tool, has been utilized to modify the genome of porcine parthenotes. However, lower biallelic mutation rate and high mosaicism frequency were observed. Here, we aimed to enhance the biallelic mutation rate with reduced mosaicism by optimization of the concentration and injection time of the Cas9/sgRNA mixture in porcine parthenotes. The results showed that the efficient biallelic mutation (93%) and low mosaicism (33%) could be achieved in porcine parthenotes by cytoplasmic injection of Cas9 mRNA/sgRNA (125/12.5 ng/μl) after 8 h of parthenogenetical activation. Thus, our study provides an effective strategy for increasing the biallelic mutation rate and population homogeneity of genetically modified parthenotes, which will strengthen the role of parthenotes in uncovering early embryonic development and assessing the mutation efficiency due to the simplicity and adaptability of CRISPR/Cas9.

Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies.

Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their αβ T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19(+) tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an "off-the-shelf" manner akin to other biologic immunopharmaceuticals

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.

The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco‐engineering and monoclonal antibody production

Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N.benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N.benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

CRISPR genome engineering and viral gene delivery: a case of mutual attraction.

The adaptation of the CRISPR/Cas9 DNA engineering machinery for mammalian cells has revolutionized our approaches to low- or high-throughput genome annotation and paved the way for conceptually novel therapeutic strategies. A large part of the attraction of CRISPR stems from the small size of its two core components--Cas9 and gRNA--and hence its compatibility with virtually any available viral vector delivery system. As a result, over the past two years, four major classes of viral vectors have already been engineered and applied as CRISPR delivery tools--retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs). The juxtaposition of these two technologies reflects a case of tremendous mutual attraction and holds unprecedented promises for biology and medicine. Here, we provide an overview of the state-of-the-art of this rapidly emerging field, from a comparative description of the principal vector designs, to a synopsis of some of the most exciting applications that were reported to date, including the use of viral CRISPR vectors for genome-wide loss-of-function screens, multiplexed gene editing or disease modeling in animals. Once specificity and safety have been improved further, viral vector-mediated in vitro/in vivo CRISPR delivery and expression promise to radically transform basic and applied biomedical research.

Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.