Multiple Gene Editing Research Articles

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori

BackgroundWith the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5′ GG motif for efficient initiation. The resulting transcripts bear a 5′ GG motif, which significantly constrains the number of targetable sites in the silkworm genome.ResultsIn this study, we used the T7 promoter to add two supernumerary G residues to the 5′ end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5′ GG mismatches depress the mutagenic activity of sgRNAs, and a single 5′ G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5′ GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5′ matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5′ GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation.ConclusionsIn summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

A SacB-based system for diverse and multiple genome editing in Gluconobacter oxydans

Gluconobacter oxydans is an important industrial bacterial strain widely used to produce a lot of useful products. However, very few gene editing tools are available for G. oxydans. This study aimed to develop an efficient genome editing method for G. oxydans using SacB as a counter-selectable marker. A plasmid that could express the kanamycin resistance gene in both E. coli and G. oxydans was constructed using the screened shuttle promoter P116. After optimizing the genome editing conditions, the derivative plasmids could be effectively utilized for diverse genome editing, such as gene deletion, insertion, replacement, and in situ modification in G. oxydans WSH-003. In addition, the SacB-based system also achieved multiple gene editing in G. oxydans. Moreover, the genome of the industrial strain G. oxydans WSH-003 was modified and the growth rate and substrate conversion rate of the strain successfully increased using this system. The system could also have potential to be applied in different G. oxydans strains. The process established in this study also provides a reference for constructing genetic tools for many other genetically recalcitrant bacteria.

Simultaneous knockout of multiple LHCF genes using single sgRNAs and engineering of a high-fidelity Cas9 for precise genome editing in marine algae.

SummaryThe CRISPR/Cas9 system is an RNA‐guided sequence‐specific genome editing tool, which has been adopted for single or multiple gene editing in a wide range of organisms. When working with gene families with functional redundancy, knocking out multiple genes within the same family may be required to generate a phenotype. In this study, we tested the possibility of exploiting the known tolerance of Cas9 for mismatches between the single‐guide RNA (sgRNA) and target site to simultaneously introduce indels in multiple homologous genes in the marine diatom Phaeodactylum tricornutum. As a proof of concept, we designed two sgRNAs that could potentially target the same six light‐harvesting complex (LHC) genes belonging to the LHCF subgroup. Mutations in up to five genes were achieved simultaneously using a previously established CRISPR/Cas9 system for P. tricornutum. A visible colour change was observed in knockout mutants with multiple LHCF lesions. A combination of pigment, LHCF protein and growth analyses was used to further investigate the phenotypic differences between the multiple LHCF mutants and WT. Furthermore, we used the two same sgRNAs in combination with a variant of the existing Cas9 where four amino acids substitutions had been introduced that previously have been shown to increase Cas9 specificity. A significant reduction of off‐target editing events was observed, indicating that the altered Cas9 functioned as a high‐fidelity (HiFi) Cas9 nuclease.

Neurodevelopmental disorders: 2021 update.

One of the current challenges in the field of neurodevelopmental disorders (NDDs) is still to determine their underlying aetiology and risk factors. NDDs comprise a diverse group of disorders primarily related to neurodevelopmental dysfunction including autism spectrum disorder (ASD), developmental delay, intellectual disability (ID), and attention-deficit/hyperactivity disorder (ADHD) that may present with a certain degree of cognitive dysfunction and high prevalence of neuropsychiatric outcomes. Last year, advances in human genomics have begun to shed light on the genetic architecture of these disorders and large-scale sequencing studies are starting to reveal mechanisms that range from unique genomic DNA methylation patterns (i.e. "episignatures") to highly polygenic conditions. In addition, the contribution of de novo somatic mutations to neurodevelopmental diseases is being recognized. However, progressing from genetic findings to underlying neuropathological mechanisms has proved challenging, due to the increased resolution of the molecular and genetic assays. Advancement in modelling tools is likely to improve our understanding of the origin of neurodevelopmental disorders and provide insight into their developmental mechanisms. Also, combined in vivo editing of multiple genes and single-cell RNA-sequencing (scRNA-seq) are bringing us into a new era of understanding the molecular neuropathology of NDDs.

CRISPR/Cas9 Systems for the Development of Saccharomyces cerevisiae Cell Factories.

Synthetic yeast cell factories provide a remarkable solution for the sustainable supply of a range of products, ranging from large-scale industrial chemicals to high-value pharmaceutical compounds. Synthetic biology is a field in which metabolic pathways are intensively studied and engineered. The clustered, regularly interspaced, short, palindromic repeat-associated (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has emerged as the state-of-the-art gene editing technique for synthetic biology. Recently, the use of different CRISPR/Cas9 systems has been extended to the field of yeast engineering for single-nucleotide resolution editing, multiple-gene editing, transcriptional regulation, and genome-scale modifications. Such advancing systems have led to accelerated microbial engineering involving less labor and time and also enhanced the understanding of cellular genetics and physiology. This review provides a brief overview of the latest research progress and the use of CRISPR/Cas9 systems in genetic manipulation, with a focus on the applications of Saccharomyces cerevisiae cell factory engineering.

Highly efficient multiplex genetic engineering of porcine primary fetal fibroblasts

BackgroundGenetically engineered porcine donors are a potential solution for the shortage of human organs for transplantation. Incompatibilities between humans and porcine donors are largely due to carbohydrate xenoantigens on the surface of porcine cells, provoking an immune response which leads to xenograft rejection. Materials and MethodsMultiplex genetic knockout of GGTA1, β4GalNT2, and CMAH is predicted to increase the rate of xenograft survival, as described previously for GGTA1. In this study, the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats–associated protein 9 system was used to target genes relevant to xenotransplantation, and a method for highly efficient editing of multiple genes in primary porcine fibroblasts was described. ResultsEditing efficiencies greater than 85% were achieved for knockout of GGTA1, β4GalNT2, and CMAH. ConclusionThe high-efficiency protocol presented here reduces scale and cost while accelerating the production of genetically engineered primary porcine fibroblast cells for in vitro studies and the production of animal models.

Amelioration of plant responses to drought under elevated CO2 by rejuvenating photosynthesis and nitrogen use efficiency: implications for future climate-resilient crops.

The contemporary global agriculture is beset with serious threats from diverse eco-environmental conditions causing decreases in crop yields by ~ 15%. These yield losses might increase further due to climate change scenarios leading to increased food prices triggering social unrest and famines. Urbanization and industrialization are often associated with rapid increases in greenhouse gases (GHGs) especially atmospheric CO2 concentration [(CO2)]. Increase in atmospheric [CO2] significantly improved crop photosynthesis and productivity initially which vary with plant species, genotype, [CO2] exposure time and biotic as well as abiotic stress factors. Numerous attempts have been made using different plant species to unravel the physiological, cellular and molecular effects of elevated [CO2] as well as drought. This review focuses on plant responses to elevated [CO2] and drought individually as well as in combination with special reference to physiology of photosynthesis including its acclimation. Furthermore, the functional role of nitrogen use efficiency (NUE) and its relation to photosynthetic acclimation and crop productivity under elevated [CO2] and drought are reviewed. In addition, we also discussed different strategies to ameliorate the limitations of ribulose-1,5-bisphosphate (RuBP) carboxylation and RuBP regeneration. Further, improved stomatal and mesophyll conductance and NUE for enhanced crop productivity under fast changing global climate conditions through biotechnological approaches are also discussed here. We conclude that multiple gene editing approaches for key events in photosynthetic processes would serve as the best strategy to generate resilient crop plants with improved productivity under fast changing climate.

Altering Plant Architecture to Improve Performance and Resistance.

High-stress resistance and yield are major goals in crop cultivation, which can be addressed by modifying plant architecture. Significant progress has been made in recent years to understand how plant architecture is controlled under various growth conditions, recognizing the central role phytohormones play in response to environmental stresses. miRNAs, transcription factors, and other associated proteins regulate plant architecture, mainly via the modulation of hormone homeostasis and signaling. To generate crop plants of ideal architecture, we propose simultaneous editing of multiple genes involved in the regulatory networks associated with plant architecture as a feasible strategy. This strategy can help to address the need to increase grain yield and/or stress resistance under the pressures of the ever-increasing world population and climate change.

Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing.

CRISPR/Cas gene editing and messenger RNA (mRNA)-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed Selective ORgan Targeting (SORT) wherein multiple classes of lipid nanoparticles (LNPs) are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen-, and liver-targeted SORT LNPs were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells, and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA / sgRNA, and Cas9 ribonucleoprotein (RNP) complexes, and is envisioned to aid development of protein replacement and gene correction therapeutics in targeted tissues.

High production of valencene in Saccharomyces cerevisiae through metabolic engineering

BackgroundThe biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application. Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction of S. cerevisiae cell factory to achieve high production of valencene will be attractive.ResultsValencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1 was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3 mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield.ConclusionsThis study achieved high production of valencene in S. cerevisiae through metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.

Simultaneous precise editing of multiple genes in human cells.

When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

A carrier-free multiplexed gene editing system applicable for suspension cells

Genetically engineered cells via CRISPR/Cas9 system can serve as powerful sources for cancer immunotherapeutic applications. Furthermore, multiple genetic alterations are necessary to overcome tumor-induced immune-suppressive mechanisms. However, one of the major obstacles is the technical difficulty with efficient multiple gene manipulation of suspension cells due to the low transfection efficacy. Herein, we established a carrier-free multiplexed gene editing platform in a simplified method, which can enhance the function of cytotoxic CD8+ T cells by modulating suspension cancer cells. Our multiple Cas9 ribonucleoproteins (RNPs) enable simultaneous disruption of two programmed cell death 1 (PD-1) ligands, functioning as negative regulators in the immune system, by accessing engineered Cas9 proteins with abilities of complexation and cellular penetration. In addition, combination with electroporation enhanced multiple gene editing efficacy, compared with that by treatment of multiple Cas9 RNPs alone. This procedure resulted in high gene editing at multiple loci of suspension cells. The treatment of multiple Cas9 RNPs targeting both ligands strongly improved Th1-type cytokine production of cytotoxic CD8+ T cells, resulting in synergistic cytotoxic effects against cancer. Simultaneous suppression of PD-L1 and PD-L2 on cancer cells via our developed editing system allows effective anti-tumor immunity. Furthermore, the treatment of multiple Cas9 RNPs targeting PD-L1, PD-L2, and TIM-3 had approximately 70–90% deletion efficacy. Thus, our multiplexed gene editing strategy endows potential clinical utilities in cancer immunotherapy.

Rapid and user-friendly open-source CRISPR/Cas9 system for single- or multi-site editing of tomato genome

CRISPR/Cas9-induced genome editing is a powerful tool for studying gene function in a variety of organisms, including plants. Using multi-sgRNAs to target one or more genes is helpful to improve the efficacy of gene editing and facilitate multi-gene editing. Here, we describe a CRISPR/Cas9 system which can be conveniently developed as a CRISPR kit. SgRNA expression cassettes can be rapidly generated by one-step PCR using our CRISPR kit. In our kit, there are two binary vectors pHNCas9 and pHNCas9HT. The binary vector pHNCas9 was constructed to allow to assemble up to eight sgRNA expression cassettes by one-step Golden Gate cloning. Another binary vector pHNCas9HT can be used to generate a large number of single target constructs by directly transforming ligation reactions products into A. tumefaciens without several procedures, such as PCR and plasmid extraction. The two binary vectors are designed according to the principles of standard BioBrick assembly to be used as an open-source tool. For example, we used BioBrick as a visual T-DNA tag. We also developed a primer design aid to complement the system. With this primer design aid, researchers can rapidly obtain primers and GC content, and sgRNA sequence of target site. Our CRISPR/Cas9 system can perform single- and multi-site editing and multiple gene editing to produce various types of mutations in tomato. This rapid and user-friendly CRISPR/Cas9 system for tomato can be potentially used for mutagenesis of important crop species for genetic improvement and is suitable for research into the function of genes.

CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium

CRISPR/Cas9 has emerged in various organisms as a powerful technology for targeted gene knockout; however, no reports of editing the Dictyostelium genome efficiently using this system are available. We describe here the application of CRISPR/Cas9-mediated gene modification in Dictyostelium. The endogenous tRNA-processing system for expressing sgRNA was approximately 10 times more effective than the commonly used U6 promoter. The resulting sgRNA affected the sub-nuclear localisation of Cas9, indicating that the expression level of sgRNA was sufficiently high to form Cas9 and sgRNA complexes within the nucleus. The all-in-one vector containing Cas9 and sgRNA was transiently expressed to generate mutants in five PI3K genes. Mutation detective PCR revealed the mutagenesis frequency of the individual genes to be between 72.9% and 100%. We confirmed that all five targeting loci in the four independent clones had insertion/deletion mutations in their target sites. Thus, we show that the CRISPR/Cas9 system can be used in Dictyostelium cells to enable efficient genome editing of multiple genes. Since this system utilises transient expression of the all-in-one vector, it has the advantage that the drug resistance cassette is not integrated into the genome and simple vector construction, involving annealing two oligo-DNAs.

CRISPR/Cas9 genome editing in wheat.

Genome editing has been a long-term challenge for molecular biology research, particularly for plants possess complex genome. The recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile tool for genome editing which enables editing of multiple genes based on the guidance of small RNAs. Even though the efficiency of CRISPR/Cas9 system has been shown with several studies from diploid plants, its application remains a challenge for plants with polyploid and complex genome. Here, we applied CRISPR/Cas9 genome editing system in wheat protoplast to conduct the targeted editing of stress-responsive transcription factor genes, wheat dehydration responsive element binding protein 2 (TaDREB2) and wheat ethylene responsive factor 3 (TaERF3). Targeted genome editing of TaDREB2 and TaERF3 was achieved with transient expression of small guide RNA and Cas9 protein in wheat protoplast. The effectiveness of mutagenesis in wheat protoplast was confirmed with restriction enzyme digestion assay, T7 endonuclease assay, and sequencing. Furthermore, several off-target regions for designed sgRNAs were analyzed, and the specificity of genome editing was confirmed with amplicon sequencing. Overall results suggested that CRISPR/Cas9 genome editing system can easily be established on wheat protoplast and it has a huge potentiality for targeted manipulation of wheat genome for crop improvement purposes.

In Vivo Genome-Wide Crispr Library Screen in a Xenograft Mouse Model of Tumor Growth and Metastasis of Multiple Myeloma

Introduction: Recent data show that Multiple Myeloma (MM) always progresses from a precursor state (monoclonal gammopathy of undetermined significance [MGUS]/smoldering multiple myeloma [SMM]) to overt MM indicating that there is continuous dissemination/clonal evolution of tumor cells from the original stages of tumor development to the time of clinical presentation. A major challenge in understanding the progression and metastasis of MM is to distinguish alterations driving the tumor growth and evolution from passenger mutations. Genetic screens are powerful tools for assaying phenotypes and identifying causal genes in various hallmarks of cancer progression. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged as a powerful technology to efficiently and simultaneously perform genome editing of multiple genes. Here we report a genome-wide CRISPR/Cas9-mediated loss of function screen in a xenograft mouse model to investigate the essential drivers of tumor growth and metastasis in MM.Methods: Lentiviral particles from 2 subpools of a human sgRNA library (Avana), each containing 1 sgRNA per gene were introduced into MM1.S (Cas9+/GFP+/Luc+) cell line with the pre-determined amount of virus to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.5-1. Cells were selected with puromycin for 5-7 days following infection to remove uninfected cells. Selected cells were injected subcutaneously into SCID-Beige mice on both flanks. Genomic DNA from pre-transplantation cells, early primary tumors (~3 weeks post tumor cell injection), late stage primary tumors and metastatic bone marrow samples were extracted. gDNA was amplified following adaptor ligation and barcoding of the samples and PCR products were subsequently sequenced on a HiSeq2000 (Illumina).Results: To investigate the sgRNA library dynamics in different sample types (pre-transplantation cells, early primary tumor, late primary tumor, and bone marrow metastasis), we compared the overall distributions of sgRNAs from all sequenced samples. The early tumor sample replicates of both subpools on average retained 77.3% and 94.7% of the sgRNAs found in the pre-transplanted cell populations, while the late primary tumors retained 59.4% and 65.6% of the sgRNAs respectively, compared to early tumors. Interestingly, only a small fraction of sgRNAs (1.1% and 3.4% of sgRNAs in the pre-transplantation cells, 10.7% and 7.2% of sgRNAs in the late primary tumors for the 2 subpools respectively) were detected in the metastatic bone marrow samples. Using gene set enrichment analysis (GSEA), we found that the gene targets of the most enriched sgRNAs in the bone marrow samples were preferentially involved in important cellular processes, such as cell cycle regulation, protein translation, and several signaling pathways. Additionally we compared sgRNAs present in early primary tumor versus pre-transplantation cells and late primary tumor and found that many sgRNAs were depleted during tumor progression, indicating that their target genes were important for progression. These depleted sgRNAs in both stages mainly targeted genes involved in mTORC1 and DNA repair pathways, many of which are regulated by MYC and cell cycle related targets of E2F transcription factors.Conclusion: We established a platform for future in vivo Cas9 screens using the genome-wide CRISPR screening libraries to explore potential new targets in regulating tumor dissemination, colonization and metastasis in MM. In addition, this in vivo screening could potentially be used to investigate essential genes of response to targeted therapies or/and immunotherapies. Thus, CRISPR/Cas9-based in vivo screening is a powerful tool for functional genomics discoveries. DisclosuresRoccaro:Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria.

203 SINGLE-STEP GENE EDITING OF 3 XENOANTIGENS IN PORCINE FIBROBLASTS USING PROGRAMMABLE NUCLEASES

Programmable nucleases (ZFN, Tal Effector Nucleases, and CRISPR) opened a new era for mammal genome editing, in particular for the pigs used for xenotransplantation. Multiple gene editing events are required both for knockout (KO) of xenoantigens and for targeted integration of human protective genes (Perota et al. 2016 J. Genet. Genomics 43, 233–23). The objective of the present work was to edit selected pig lines to KO the enzymes coding for the most relevant xenoantigens (i.e. GGTA1, CMAH, and B4GalNT2), combining Talens and CRISPR/Cas9 technologies to magnetic beads selection (Li et al. 2013 Xenotransplantation 22, 20–31). Primary porcine adult fibroblasts were transfected using Nucleofector (V-024 program). In a single reaction 2 × 106 fibroblasts were co-transfected using 2 different sets of TALENS (4 μg/set) specific for CMAH (Conchon et al., 2013) and GGTA1 (Perota et al., 2015) genes together with B4GalNT2-specific CRISPR/Cas9 expression vector (2 μg; pX330-B4GalNT2; Estrada et al., 2015). Eight days post-transfection (DPT), Gal–/– cells were selected initially using biotin-conjugated IB4 lectin (Sigma, St. Louis, MO, USA) and magnetic beads (Dynabeads M-280, Thermo Fisher Scientific, Waltham, MA, USA). The selected cells were then plated on 150-mm Petri dishes (200 cells/dish) and cultured for 10 days. Selected colonies were expanded for PCR analysis and cryopreserved for somatic cell nuclear transfer (SCNT). All colonies were analysed by PCR for CMAH gene and their resulting products were digested with HindIII (HindIII-RFLP). Colonies that lost wild-type HindIII as a consequence of Talens effected deletion were PCR characterised for GGTA1, selecting those that had detectable Indels after gel electrophoresis and finally analysed by PCR for B4GalNT2. All PCR products were validated by sequencing for all the 3 genes of interest (TopoTA, Thermo Fisher Scientific). Selected colonies were used as nuclear donors for SCNT (Lagutina et al., 2006). Eight DPT we obtained 3.45 ×106 cells. About 6.0 × 103 Gal-negative cells (0.17%) were collected from the supernatant after magnetic beads separation. Eighteen DPT, 120 colonies were picked up and their HindIII-RFLP analyses on CMAH gene revealed that 22 colonies (18.3%) were KO for both CMAH alleles. Of these 22 colonies following electrophoretic analyses of GGTA1-PCR products, 13 colonies had detectable Indels. These 13 colonies were finally PCR analysed and sequenced for B4GalNT2 and sequenced. Final sequencing results confirmed that 2 colonies (1.6%) resulted in KO for the 3 genes. Three different zona-free SCNT experiments were done and 579 reconstructed embryos were obtained. On Day 7, 322 morulae or blastocysts (56%) were transferred in 3 synchronised sows and 2 (66%) became pregnant. In conclusion, after gene editing with programmable nucleases, combining beads-mediated selection with well-designed molecular analyses, we developed a multistep assay that can be used efficiently to detect desired gene edited events in cell colonies suitable for the SCNT. Embryos generated after SCNT were able to establish pregnancies at a high rate. This work is supported by European FP7 grants Translink (n° 603049) and Xenoislet (n° 601827).

A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection–based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create “Cas9 transgene-free” gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice.

A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis.

The recently developed CRISPR/Cas9 system is a promising technology for targeted genome editing in a variety of species including plants. However, the first generation systems were designed to target one or two gene loci at a time. We designed a new multiplex CRISPR/Cas9 system that allows the co-expression of six sgRNA modules in one binary vector using a simple (three steps) cloning strategy in Arabidopsis. The transcription of the sgRNA modules is under the control of three different RNA Polymerase III-dependent promoters. We tested the efficiency of the new multiplex system by targeting six of the fourteen PYL families of ABA receptor genes in a single transformation experiment. One line with mutations in all six targeted PYLs was identified from 15 T1 plants. The mutagenesis frequency for the six individual PYL targets in the T1 lines ranged from 13 to 93%. In the presence of ABA, the transgenic line identified as containing mutations in all six PYL genes produced the highest germination rate in the T2 progeny (37%). Among these germinated seedlings, half of the analyzed plants (15/30) were homozygous mutants for at least four targeted genes and two plants (6.7%) contained homozygous mutations in five of the targeted PYLs and the other targeted PYL had biallelic mutations. Homozygous sextuple mutants were identified in the T3 progeny and characterized together with previously described triple and sextuple PYL mutants. We anticipate that the application of this multiplex CRISPR/Cas9 system will strongly facilitate functional analysis of genes pathways and families.

Multiplex Cripsr/Cas9 Genome Editing to Generate Potent Universal CART and PD1-Deficient Cells Against Leukemia

Engineered CAR T (CART) cell treatments of cancer patients have shown promising results. The majority of current CART clinical trials utilizes autologous T cells and might therefore be hampered by the poor quality and quantity of T cells as well as the time and expense of manufacturing autologous T cell products. These limitations would be circumvented by the use of allogeneic T cells. However, the endogenous TCR on allogeneic T cells may recognize the alloantigens of the recipient, leading to graft-versus-host disease (GVHD); furthermore, the expression of HLA on the surface of allogeneic T cells causes rapid rejection by the host immune system. Therefore, simple and efficient methods are needed for multiplex genomic editing of T cells. The CRISPR/Cas9 system has recently emerged as a potentially robust alternative for inducing targeted genetic alterations and as a process for multiplex genome engineering.In the present study, by using CRISPR/Cas9 system to simultaneously disrupt multiple genomic loci. we have generated CART cells deficient in the expression of endogenous TCR, HLA class I (HLA-I) and PD1 that can be used as allogeneic universal CART cells. We found that TCR α chain, TCR β chain, beta-2 microglobulin (B2M) and PD1 genes could be disrupted with high efficiency through the co-introduction of mRNA encoding the Cas9 with gRNAs targeting these genes by electroporation. We observed over 80% percent gene ablation at protein levels by single gene disruption and over 70% percent of TCR and HLA-I double ablation by multiplex gene disruption.Universal PD1 deficient CD19 CART cells could be generated by integrating this multiple gene editing technology into to the standard clinical CD19 CAR lentiviral transduction procedure and expanded to the clinical scale. The TCR/HLA-I double-negative CD19 CART showed robust in vitro anti-tumor activities, such as lytic capacity, cytokine secretion and proliferation, as potent as those of the wild-type CD19 CART cells. The T cells were tested in NSG mice bearing disseminated Nalm6 leukemia. Mice treated with CD19 CART cells with endogenous TCR disrupted or with a simultaneous disruption of TCR and HLA-I exhibited tumor regression similar to that of mice treated with wild-type CD19 CART cells, suggesting that the disruption of TCR alone or together with B2M does not affect CART cell anti-tumor activity. In addition, we found that simultaneous disruption of endogenous PD1 enhances the efficacy of the universal CD19 CART therapy in vivo in a Nalm6-PDL1 leukemia model.The universal T cells we generated showed reduced alloreactivity, demonstrated in a one-way MLR using an IFNg Elispot assay when incubated with irradiated allogeneic PBMCs. The GVHD effect of the engineered T cells was also tested in different NSG models. In a Nalm6 leukemia NSG model, the mice treated with the double or triple knock out CAR T cells did not develop any signs of GVHD. By contrast, 3 out of 4 mice from the wild-type CD19 CART group developed xenogeneic GVHD at day 65, which was confirmed by histological examination of different organs. In another experiment, T cells were infused intravenously into sub-lethally irradiated mice. GVHD was monitored 2 to 3 times per week, and 4 out 5 mice receiving wild type T cell developed GVHD, while PBS treated, TCR single and TCR/HLA-I double ablated T cell treated group did not show any signs of GVHD.In summary clinical scale universal CD19 CART cells, with potent anti-leukemia activity and reduced alloreactivity can be efficiently generated using multiplex CRISPR/Cas9 technology. This approach can be incorporated into current GMP-compliant manufacturing procedures and has a high potential for translation, and thus provides an alternative to autologous CART cells. DisclosuresRen:University of Pennsylevania: Patents & Royalties. Liu:University of Pennsylevania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Zhao:Novartis: Patents & Royalties, Research Funding.