Cas9 Ribonucleoprotein Complexes Research Articles

A Streamlined Protocol for Wheat (Triticum aestivum) Protoplast Isolation and Transformation With CRISPR-Cas Ribonucleoprotein Complexes.

The genetic engineering method CRISPR has been touted as an efficient, inexpensive, easily used, and targeted genetic modification technology that is widely suggested as having the potential to solve many of the problems facing agriculture now and in the future. Like all new technologies, however, it is not without challenges. One of the most difficult challenges to anticipate and detect is gene targets that are inaccessible due to the chromatin state at their specific location. There is currently no way to predict this during the process of designing a sgRNA target, and the only way to detect this issue before spending time and resources on full transformations is to test the cleavage ability of the sgRNA in vivo. In wheat, this is possible using protoplast isolation and PEG transformation with Cas9 ribonucleoprotein complexes. Therefore, we have developed a streamlined protocol for testing the accessibility of sgRNA targets in wheat. The first steps involve digesting wheat leaf tissue in an enzymatic solution and then isolating viable protoplasts using filters and a sucrose gradient. The protoplasts are then transformed using Cas9 ribonucleoprotein complexes via PEG-mediated transformation. DNA is isolated from the CRISPR-Cas-edited protoplasts and PCR is performed to amplify the gene target region. The PCR product is then used to assess the editing efficiency of the chosen sgRNA using Sanger sequencing. This simplified protocol for the isolation and transformation of wheat protoplast cells using Cas9 ribonucleoprotein complexes streamlines CRISPR transformation projects by allowing for a fast and easy test of sgRNA accessibility in vivo.

Enabling the advancement of non-viral CRISPR-based cell therapy with a new Large-scale electroporation platform

Background & Aim Crispr-Cas9 based gene modification is gaining traction in the field of cell-based immunotherapy as a safe non-viral alternative to lentiviral vectors which are currently utilized to manufacture the approved FDA Car-T therapies. However, the performance benefit and specificity of the Cas-9 protein can only be realized with a delivery platform that can efficiently and safely deliver the ribonucleoprotein complex (RNP) to the target cells. In addition, with the speed at which ex vivo modified cell-therapies are moving into process development and manufacturing phases, it is imperative to not only achieve performance targets but also be able to quickly integrate into regulated and controlled environments. Methods, Results & Conclusion A newly developed large-scale electroporation platform has shown efficient gene editing in human primary t-cells. Targeting multiple genes, to show the flexibility of the platform, knock-out of the target loci was achieved reproducibly and in a scalable manner from small scale to large scale. In addition, newly developed knock-in enhancers, are able to achieve knock-in efficiency with a single electroporation delivering the Cas9 RNP and donor DNA all at once. Data will be presented to show the integration efficiency of various size donor DNA constructs at multiple loci with primary t-cells that have been isolated from fresh leukapheresis, activated with CD3/CD28 Dynabeads™, cultured with OpTmizer™ media and electroporated 3-days post activation. The promising results of gene delivery and cell engineering in primary T-cells are helping to advance new platforms and cell therapies forward with unprecedent speed. The technological advancement of CRISPR-Cas9 in combination with electroporation is able to provide researchers and drug developers the ability to quickly and seamlessly scale their therapeutic platforms from early research and development to clinical manufacturing. Speed, efficiency and proper quality control are critical to ensure that more patients are able to benefit from the promising cell and gene therapies being developed. Crispr-Cas9 based gene modification is gaining traction in the field of cell-based immunotherapy as a safe non-viral alternative to lentiviral vectors which are currently utilized to manufacture the approved FDA Car-T therapies. However, the performance benefit and specificity of the Cas-9 protein can only be realized with a delivery platform that can efficiently and safely deliver the ribonucleoprotein complex (RNP) to the target cells. In addition, with the speed at which ex vivo modified cell-therapies are moving into process development and manufacturing phases, it is imperative to not only achieve performance targets but also be able to quickly integrate into regulated and controlled environments. A newly developed large-scale electroporation platform has shown efficient gene editing in human primary t-cells. Targeting multiple genes, to show the flexibility of the platform, knock-out of the target loci was achieved reproducibly and in a scalable manner from small scale to large scale. In addition, newly developed knock-in enhancers, are able to achieve knock-in efficiency with a single electroporation delivering the Cas9 RNP and donor DNA all at once. Data will be presented to show the integration efficiency of various size donor DNA constructs at multiple loci with primary t-cells that have been isolated from fresh leukapheresis, activated with CD3/CD28 Dynabeads™, cultured with OpTmizer™ media and electroporated 3-days post activation.

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.

Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control.

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.

Targeted somatic mutagenesis through CRISPR/Cas9 ribonucleoprotein complexes in the olive fruit fly, Bactrocera oleae.

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo-we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene-specific single-guide RNAs. Gene disruption of Bo-we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo-we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.

An engineered exosome for delivering sgRNA:Cas9 ribonucleoprotein complex and genome editing in recipient cells.

CRISPR-Cas9 is a versatile genome-editing technology that is a promising gene therapy tactic. However, the delivery of CRISPR-Cas9 is still a major obstacle to its broader clinical application. Here, we confirm that the components of CRISPR-Cas9-sgRNA and Cas9 protein-can be packaged into exosomes, where sgRNA and Cas9 protein exist as a sgRNA:Cas9 ribonucleoprotein complex. Although exosomal CRISPR-Cas9 components can be delivered into recipient cells, they are not adequate to abrogate the target gene in recipient cells. To solve this, we engineered a functionalized exosome (M-CRISPR-Cas9 exosome) that could encapsulate CRISPR-Cas9 components more efficiently. To improve the loading efficiency of Cas9 proteins into exosomes, we artificially engineered exosomes by fusing GFP and GFP nanobody with exosomal membrane protein CD63 and Cas9 protein, respectively. Therefore, Cas9 proteins could be captured selectively and efficiently loaded into exosomes due to the affinity of GFP-GFP nanobody rather than random loading. sgRNA and Cas9 protein exist as a complex in functionalized exosomes and can be delivered into recipient cells. To show the function of modified exosomes-delivered CRISPR-Cas9 components in recipient cells visually, we generated a reporter cell line (A549stop-DsRed) that produced a red fluorescent signal when the stop element was deleted by the sgRNA-guided endonuclease. Using A549stop-DsRed reporter cells, we showed that modified exosomes loaded with CRISPR-Cas9 components abrogated the target gene more efficiently in recipient cells. Our study reports an alternative tactic for CRISPR-Cas9 delivery.

Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.

Specific Gene Disruption in the Major Livestock Pests Cochliomyia hominivorax and Lucilia cuprina Using CRISPR/Cas9.

Cochliomyia hominivorax and Lucilia cuprina are major pests of livestock. Their larvae infest warm-blooded vertebrates and feed on host’s tissues, resulting in severe industry losses. As they are serious pests, considerable effort has been made to develop genomic resources and functional tools aiming to improve their management and control. Here, we report a significant addition to the pool of genome manipulation tools through the establishment of efficient CRISPR/Cas9 protocols for the generation of directed and inheritable modifications in the genome of these flies. Site-directed mutations were introduced in the C. hominivorax and L. cuprina yellow genes (ChY and LcY) producing lightly pigmented adults. High rates of somatic mosaicism were induced when embryos were injected with Cas9 ribonucleoprotein complexes (RNPs) pre-assembled with guide RNAs (sgRNAs) at high concentrations. Adult flies carrying disrupted yellow alleles lacked normal pigmentation (brown body phenotype) and efficiently transmitted the mutated alleles to the subsequent generation, allowing the rapid creation of homozygous strains for reverse genetics of candidate loci. We next used our established CRISPR protocol to disrupt the C. hominivorax transformer gene (Chtra). Surviving females carrying mutations in the Chtra locus developed mosaic phenotypes of transformed ovipositors with characteristics of male genitalia while exhibiting abnormal reproductive tissues. The CRISPR protocol described here is a significant improvement on the existing toolkit of molecular methods in calliphorids. Our results also suggest that Cas9-based systems targeting Chtra and Lctra could be an effective means for controlling natural populations of these important pests.

Cas9 Ribonucleoprotein Complex Delivery: Methods and Applications for Neuroinflammation.

The CRISPR/Cas9 system is a revolutionary gene editing technology that combines simplicity of use and efficiency of mutagenesis. As this technology progresses toward human therapies, valid concerns including off-target mutations and immunogenicity must be addressed. One approach to address these issues is to minimize the presence of the CRISPR/Cas9 components by maintaining a tighter temporal control of Cas9 endonuclease and reducing the time period of activity. This has been achieved to some degree by delivering the CRISPR/Cas9 system via pre-formed Cas9 + gRNA ribonucleoprotein (RNP) complexes. In this review, we first discuss the molecular modifications that can be made using CRISPR/Cas9 and provide an overview of current methods for delivering Cas9 RNP complexes both in vitro and in vivo. We conclude with examples of how Cas9 RNP delivery may be used to target neuroinflammatory processes, namely in regard to viral infections of the central nervous system and neurodegenerative diseases. We propose that Cas9 RNP delivery is a viable approach when considering the CRISPR/Cas9 system for both experimentation and the treatment of disease. Graphical Abstract.

S852 IN VIVO SELECTION FOR CORRECTED BETA‐GLOBIN ALLELES AFTER CRISPR/CAS9 EDITING IN HUMAN SICKLE HEMATOPOIETIC STEM CELLS ENHANCES THERAPEUTIC POTENTIAL

Background:Sickle Cell Disease (SCD) is a devastating disorder caused by a single base change in the ß‐globin gene (HBB) that is one component of the adult hemoglobin tetramer (HbA, which contains 2 a‐globin and 2 ß‐globin proteins). SCD is a recessive disorder – if one of the 2 alleles of HBB is normal, the disease is not present; thus gene correction needs to correct only one of the two HBB alleles in any given stem cell.Aims:To correct the sickle allele in human hematopoietic stem cellsMethods:To correct the sickle mutation, we developed a process that directly introduces a preassembled Cas9 ribonucleoprotein (RNP) complex (composed of recombinant Cas9 protein and single guide RNA) along with a single‐stranded DNA oligonucleotide donor template (ssODN) for homology‐directed repair (HDR). We studied gene correction after long‐term xenografting of human sickle HSCs. After electroporation with RNP and ssODN, sickle human HSCs were injected into immunodeficient NBSGW mice, which permit engraftment of human HSCs and erythroid differentiation in the bone marrow.Results:Sixteen to 20 weeks after injection, we analyzed engrafted human cells in the bone marrow of 43 mice in 4 cohorts. We quantified editing in xenografted cells, and found an average of 23.4% of HBB alleles with the corrected genotype, and 65.2% with insertion/deletion mutations (indels). To assess the distribution of HBB alleles within populations of xenografted cells, we inferred HBB genotypes using RNA‐Seq data from 359 individual clonal BFU‐E colonies derived from marrow CD34+ cells (figure 1A). This reveals that a substantial proportion of corrected (HDR) alleles are heterozygous with indel alleles equivalent to ß‐thalassemia mutations, confirming that corrected (wild type) HBB alleles in edited HSCs are distributed such that many erythrocytes have only a single wild type HBB allele. Data from the colony analysis, extrapolated to the entire experimental group, indicates that the 23.4% of corrected HBB alleles are distributed among >30% of HSCs, producing a comparable proportion of functional erythrocytes. SCD is a recessive disorder; based upon observations of mixed chimerism after allogeneic transplantation, this proportion of normal cells is consistent with cure.From engrafted NBSGW mice, we immunoselected human erythroid cells with anti‐CD235a (GlycophorinA), carried out RNA‐Seq, and inferred HBB genotypes in the erythroblast population. For each individual mouse, we compared the proportion of corrected HBB alleles in the xenografted bone marrow, in CD34+ cells immunoselected from marrow, and in CD235a+ erythroblasts. To control for enrichment of alleles corrected by HDR, we distinguished two types of HDR: “PAM‐only” HDR events mutate the Cas9 PAM without the conversion tract reaching the sickle mutation, whereas “sickle‐corrected” HDR events mutate the Cas9 PAM and also correct the sickle mutation. We observe a marked enrichment of “sickle‐corrected” HDR alleles in erythroblasts when compared to marrow or CD34+ cells from the same mouse, but no enrichment of “PAM‐only” HDR alleles (figure 1B).Summary/Conclusion:In summary, we have developed a Cas9‐mediated gene editing protocol that reproducibly yields a cell product in which the sickle mutation is corrected to wild type at a level that is sufficient to cure SCD. Evidence we have presented indicates that cells carrying the corrected ß‐globin gene are enriched during in vivo erythroid differentiation. These results support moving this therapeutic approach to the clinic.image

Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.

Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes

BackgroundThe CRISPR/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes. Established tools for many fungal species exist while most of them are based on in vivo expressed Cas9 and guide RNA (gRNA). Alternatively, in vitro assembled Cas9 and gRNA ribonucleoprotein complexes can be used in genome editing, however, only a few examples have been reported in fungi. In general, high-throughput compatible transformation workflows for filamentous fungi are immature.ResultsIn this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger strain with improved capacity to produce galactarate, a potential chemical building block, was generated.ConclusionsThe developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. In addition, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.

Targeted Gene Disruption in Pacific Oyster Based on CRISPR/Cas9 Ribonucleoprotein Complexes.

The Pacific oyster (Crassostrea gigas) is a representative bivalve mollusc that is widely cultured in the world. In recent years, it has become an important model species for ecological, evolutionary, and developmental studies because of its ability to survive in extreme environmental conditions as a sessile filter feeder and its classical mosaic pattern of development. Although the complete genome sequence of C. gigas is available and omics data have been rapidly generated for the past few years, the genetic tools for gene functional studies have thus far been limited to RNA interference technology. In this study, we developed a gene editing system for C. gigas based on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 ribonucleoprotein complexes. Two candidate genes, myostatin (MSTN) and Twist, were selected as targets. After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, CRISPR-induced indel mutations were detected in the target genes. The CRISPR/Cas9-induced mutations were predominantly small indel mutations ranging in size from 1 to 24bp in these two target genes. These results demonstrate that CRISPR/Cas9 can be successfully used as an effective targeted gene editing system in C. gigas. The method reported here provides a powerful tool for gene functional studies in oysters and other marine bivalves, and potentially as a new technology for genetic engineering to improve oyster traits for aquaculture.

Efficient Intracellular Delivery of CRISPR-Cas Ribonucleoproteins through Receptor Mediated Endocytosis.

We recently reported a new delivery system harnessing surface receptors for targeted uptake of CRISPR-Cas9 ribonucleoprotein into mammalian cells (Rouet et al., JACS 2018). For this purpose, Cas9 protein was labeled with the small molecule ligand ASGRL, specific for the asialoglycoprotein receptor, enabling endosomal uptake of the ribonucleoprotein into human cells expressing the receptor. However, detailed mechanistic insights had remained unknown and editing efficiency low. Here we investigate the mechanism of endosomal escape as mediated by the ppTG21 endosomolytic peptide and outline the development of novel Cas9 or Cas12a ribonucleoprotein complexes with increased editing efficiency.

204 Efficient editing of porcine parthenogenetic zygotes by electroporation of Cas9 ribonucleoprotein complexes

Gene editing by microinjection is an efficient system to produce mutant livestock; however, microinjection is time-consuming and requires special skill, limiting its use for large-scale production of gene-edited animals. Therefore, the aim of this study was to develop a system to deliver guide (g)RNA/Cas9/ribonucleoprotein (RNP) by electroporation into parthenogenic porcine zygotes. For experiment 1, we delivered gRNA/Cas9 RNP (250ng μL−1 of each), targeting GATA4 using 2 electroporation conditions. Group 1 (n=130): 20V, 3ms, ×2 pulses, 1 repeat; group 2 (n=102): 20V, 1ms, ×2 pulses, 2 repeats; and Control (n=96): parthenogenic zygotes, no electroporation. For experiment 2, we delivered gRNA/Cas9 RNP (250ng μL−1 of each) targeting ROSA26 by electroporation with 4 conditions compared with delivery of RNP by microinjection: group 1 (n=17): 20V, 3ms, ×1 pulses, 1 repeat; group 2 (n=49): 20V, 3ms, ×3 pulses, 1 repeat; group 3 (n=64): 30V, 3ms, ×1 pulses, 1 repeat; group 4 (n=61): 30V, 3ms, ×3 pulses, 1 repeat; group 5 (n=120): zygotes microinjected with Cas9/ROSA26 sgRNA (25/25ng μL−1), and Control (n=76): parthenogenic zygotes, no electroporation. The electroporated zygotes were cultured in porcine zygote medium-3 (PZM-3) with controlled atmosphere, and development was evaluated on Day 2 (cleavage) and Day 7 (blastocyst rate). Gene editing was evaluated on embryos (blastocyst and morulas) by PCR and Sanger sequencing of amplicons including the RNP target site. Data were compared using chi-squared test, and differences were considered significant at P<0.05. Cleavage rates in experiment 1 were similar for the control (86/96; 89.5%), group 1 (94/102; 92.1%), and group 2 (119/130; 91.5%). Blastocyst rates were higher for the control (46/96; 47%) than for the other groups (P<0.01). However, for the treated groups, the blastocyst rates were similar, group 1 (19/102; 9.2%) and group 2 (12/130; 18.6%). Furthermore, the non-homologous end joining (NHEJ) efficiency was similar for groups 1 (14/18; 77.7%) and 2 (14/17; 82.3%). In experiment 2, the cleavage (53/76; 69%) and blastocyst rates (30/76; 39%) were significantly higher for the control than for the treated groups (P<0.01). Among the groups, the lower cleavage and blastocyst rates were for group 4 (20/61; 32.7% and 3/61; 4.9%, respectively) compared with the other electroporation and microinjection groups (P<0.03). However, NHEJ efficiency was higher for electroporation groups 2 (6/8; 75%), 3 (17/17; 100%), and 4 (2/2; 100%) compared with microinjection (2/15; 13%). In conclusion, electroporation of Cas9/RNP is an efficient alternative to microinjection for gene editing in porcine zygotes.

TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.

Cas9 ribonucleoprotein complex allows direct and rapid analysis of coding and noncoding regions of target genes in Pleurodeles waltl development and regeneration

Newts have remarkable ability to regenerate their organs and have been used in research for centuries. However, the laborious work of breeding has hampered reverse genetics strategies in newt. Here, we present simple and efficient gene knockout using Cas9 ribonucleoprotein complex (RNP) in Pleurodeles waltl, a species suitable for regenerative biology studies using reverse genetics. Most of the founders exhibited severe phenotypes against each target gene (tyrosinase, pax6, tbx5); notably, all tyrosinase Cas9 RNP-injected embryos showed complete albinism. Moreover, amplicon sequencing analysis of Cas9 RNP-injected embryos revealed virtually complete biallelic disruption at target loci in founders, allowing direct phenotype analysis in the F0 generation. In addition, we demonstrated the generation of tyrosinase null F1 offspring within a year. Finally, we expanded this approach to the analysis of noncoding regulatory elements by targeting limb-specific enhancer of sonic hedgehog, known as the zone of polarizing activity regulatory sequence (ZRS; also called MFCS1). Disruption of ZRS led to digit deformation in limb regeneration. From these results, we are confident that this highly efficient gene knockout method will accelerate gene functional analysis in the post-genome era of salamanders.

High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion

Targeted mutagenesis using CRISPR/Cas9 technology has been shown to be a powerful approach to examine gene function in diverse metazoan species. One common drawback is that mixed genotypes, and thus variable phenotypes, arise in the F0 generation because incorrect DNA repair produces different mutations amongst cells of the developing embryo. We report here an effective method for gene knockout (KO) in the hydrozoan Clytia hemisphaerica, by injection into the egg of Cas9/sgRNA ribonucleoprotein complex (RNP). Expected phenotypes were observed in the F0 generation when targeting endogenous GFP genes, which abolished fluorescence in embryos, or CheRfx123 (that codes for a conserved master transcriptional regulator for ciliogenesis) which caused sperm motility defects. When high concentrations of Cas9 RNP were used, the mutations in target genes at F0 polyp or jellyfish stages were not random but consisted predominantly of one or two specific deletions between pairs of short microhomologies flanking the cleavage site. Such microhomology-mediated (MM) deletion is most likely caused by microhomology-mediated end-joining (MMEJ), which may be favoured in early stage embryos. This finding makes it very easy to isolate uniform, largely non-mosaic mutants with predictable genotypes in the F0 generation in Clytia, allowing rapid and reliable phenotype assessment.

Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing

Cas9-mediated gene editing is a powerful tool for addressing research questions in arthropods. Current approaches rely upon delivering Cas9 ribonucleoprotein (RNP) complex by embryonic microinjection, which is challenging, is limited to a small number of species, and is inefficient even in optimized taxa. Here we develop a technology termed Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to deliver Cas9 RNP to the arthropod germline by injection into adult female mosquitoes. We identify a peptide (P2C) that mediates transduction of Cas9 RNP from the female hemolymph to the developing mosquito oocytes, resulting in heritable gene editing of the offspring with efficiency as high as 0.3 mutants per injected mosquito. We demonstrate that P2C functions in six mosquito species. Identification of taxa-specific ovary-specific ligand–receptor pairs may further extend the use of ReMOT Control for gene editing in novel species.

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins.

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGFβ. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.