Cas9 mRNA Research Articles

Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression

Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Recently, virus-like particles (VLPs) were developed for mRNA or ribonucleoprotein (RNP) delivery for short-term endonuclease expression. Generating large amount of LVs or VLPs is challenging. On the other hand, methods for using VLPs to co-deliver Cas9 mRNA and single guide RNA (sgRNA) are limited. Fusing aptamer-binding protein (ABP) to the N-terminus of HIV Gag protein is currently the successful way to develop hybrid particles for co-delivering Cas9 mRNA and sgRNA. The effects of modifying Gag protein this way on particle assembly are unknown. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on these observations, here we developed hybrid particles for Cas9 mRNA and sgRNA co-delivery with normal capsid assembly efficiency. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral genomic RNA, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, here we describe the development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA, and new LVs for enhanced vector production and gene expression.

Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors.

CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'-O-methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'-O-methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.

Optimization of CRISPR/Cas9-mediated gene disruption in Xenopus laevis using a phenotypic image analysis technique.

The CRISPR/Cas9 method has become popular for gene disruption experiments in Xenopus laevis. However, the experimental conditions that influence the efficiency of CRISPR/Cas9 remain unclear. To that end, we developed an image analysis technique for the semi-quantitative evaluation of the pigment phenotype resulting from the disruption of tyrosinase genes in X. laevis using a CRISPR/Cas9 approach, and then examined the effects of varying five experimental parameters (timing of the CRISPR reagent injection into developing embryos; amount of Cas9 mRNA in the injection reagent; total injection volume per embryo; number of injection sites per embryo; and the culture temperature of the injected embryos) on the gene disruption efficiency. The results of this systematic analysis suggest that the highest possible efficiency of target gene disruption can be achieved by injecting a total of 20 nL of the CRISPR reagent containing 1500 pg of Cas9 mRNA or 4 ng of Cas9 protein into two separate locations (10nL each) of one-cell stage embryos cultured at 22°C. This study also highlights the importance of balancing the experimental parameters for increasing gene disruption efficiency and provides valuable insights into the optimal conditions for applying the CRISPR/Cas9 system to new experimental organisms.

Effects of the ITGA2B Nonsense Mutation (c.2659C > T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology

To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.

KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export

CRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. Previous studies have revealed the feasibility of modulating Cas9-based genome- and base-editing tools using protein or small-molecule CRISPR inhibitors. Here we screened a set of small molecule compounds with irreversible warhead, aiming to identifying small-molecule modulators of CRISPR-Cas9. It was found that selective inhibitors of nuclear export (SINEs) could efficiently inhibit the cellular activity of Cas9 in the form of genome-, base- and prime-editing tools. Interestingly, SINEs did not function as direct inhibitors to Cas9, but modulated Cas9 activities by interfering with the nuclear export process of Cas9 mRNA. Thus, to the best of our knowledge, SINEs represent the first reported indirect, irreversible inhibitors of CRISPR-Cas9. Most importantly, an FDA-approved anticancer drug KPT330, along with other examined SINEs, could improve the specificities of CRISPR-Cas9-based genome- and base editing tools in human cells. Our study expands the toolbox of CRISPR modulating elements and provides a feasible approach to improving the specificity of CRISPR-Cas9-based genome engineering tools.

Influence of Hydrophobicity in the Hydrophilic Region of Cationic Lipids on Enhancing Nucleic Acid Delivery and Gene Editing.

Intracellular delivery of biomolecules using non-viral vectors critically depends on the vectors' ability to allow the escape and release of the contents from the endosomes. Prior findings demonstrated that aromatic/hydrophobic group-containing amino acids such as phenylalanine (F) and tryptophan (W) destabilize cellular membranes by forming pores in the lipid bilayer. Taking cues from these findings, we have developed four α-tocopherol-based cationic amphiphiles by varying the aromatic/hydrophobic amino acids such as glycine (G), proline (P), phenylalanine (F), and tryptophan (W) as head groups and triazole in the linker region to study their impact on endosomal escape for the enhanced transfection efficacy. The lipids tocopherol-triazole-phenylalanine (TTF) and tocopherol-triazole-tryptophan (TTW) exhibited similar potential to commercial transfecting reagents, Lipofectamine (LF) 3000 and Lipofectamine Messenger Max (LFMM), respectively, in transfecting plasmid DNA and messenger RNA in multiple cultured cell lines. The TTW liposome was also found to be effective in delivering Cas9 mRNA and demonstrated equal efficiency of gene editing AAVS1 locus compared to LFMM in CHO, Neuro-2a, and EA.HY926 cell lines. In this current investigation, it is shown that the synthesized cationic lipids with aromatic hydrophobic R group-containing amino acids are safe, economic, and actually more efficient in nucleic acid delivery and genome-editing applications. These findings can be further explored in the genome-editing approach for treating genetic disorders.

Key sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9.

Specific sequence features of the protospacer and protospacer-adjacent motif (PAM) are critical for efficient cleavage by CRISPR-Cas9, but current knowledge is largely derived from single-guide RNA (sgRNA) systems assessed in cultured cells. In this study, we sought to determine gRNA sequence features of a more native CRISPR-Cas9 ribonucleoprotein (RNP) complex with dual-guide RNAs (dgRNAs) composed of crRNA and tracrRNA, which has been used increasingly in recent CRISPR-Cas9 applications, particularly in zebrafish. Using both wild-type and HiFi SpCas9, we determined on-target cleavage efficiencies of 51 crRNAs in zebrafish embryos by assessing indel occurrence. Statistical analysis of these data identified novel position-specific mononucleotide features relevant to cleavage efficiencies throughout the protospacer sequence that may be unique to CRISPR-Cas9 RNPs pre-assembled with perfectly matched gRNAs. Overall features for wild-type Cas9 resembled those for HiFi Cas9, but specific differences were also observed. Mutational analysis of mononucleotide features confirmed their relevance to cleavage efficiencies. Moreover, the mononucleotide feature-based score, CRISPR-kp, correlated well with efficiencies of gRNAs reported in previous zebrafish RNP injection experiments, as well as independently tested crRNAs only in RNP format, but not with Cas9 mRNA co-injection. These findings will facilitate design of gRNA/crRNAs in genome editing applications, especially when using pre-assembled RNPs.

Size Exclusion and Ion Exchange Chromatographic Hardware Modified with a Hydrophilic Hybrid Surface.

Certain biomolecules have proven to be difficult to analyze by liquid chromatography (LC), especially under certain chromatographic conditions. The separation of proteins in aqueous mobile phases is one such example because there is the potential for both hydrophobic and ionic secondary interactions to occur with chromatographic hardware to the detriment of peak recovery, peak shape, and the overall sensitivity of the LC analysis. To decrease non-specific adsorption and undesired secondary interactions between column hardware and biomolecules, we have developed and applied a new hydrophilically modified hybrid surface (h-HST) for size exclusion chromatography (SEC) and anion exchange (AEX) separations of proteins and nucleic acids. This surface incorporates additional oxygen and carbon atoms onto an ethylene bridge hybrid siloxane polymer. As a result, it exhibits reduced electrostatic properties and hydrophilicity that facilitates challenging aqueous separations. Flow injection tests with a phosphate buffer showed superior protein recovery from an h-HST frit when compared to unmodified ethylene-bridged hybrid HST, titanium, stainless steel, and PEEK frits. When applied to SEC of rituximab, ramucirumab, and trastuzumab emtansine with a 50 mM ammonium acetate buffer, this new hydrophilic chromatographic hardware yielded improved monomer and aggregate recovery, higher plate numbers, and more symmetrical peaks. AEX columns also benefited from h-HST hardware. An acidic mAb (eculizumab) showed improved recovery, more stable retention, and a sharper peak when eluted from an h-HST versus SS column. Moreover, AEX separations of intact mRNA samples (Cas9 and EPO mRNA) were improved, where it was seen that h-HST column hardware provided higher sensitivity and more repeatable peak areas from injection to injection. As such, there is significant potential in the use of h-HST chromatographic hardware to facilitate more robust and more sensitive analyses for a multitude of challenging separations and analytes.

Development of an RNA anti-parasite based on CRISPR-Cas9 against Chilodonella piscicola

Chilodonella piscicola is a gill and body surface parasite of freshwater fish, affecting the growth of juvenile and adult freshwater fishes. To develop an effective method to manage C.piscicola in aquaculture, we used CRISPR/Cas9 technology to design and validate small guide RNA targeting DNA barcoding ITS-1 and 18S rDNA sequences of C.piscicola to develop a species-specific pesticide. In this study, effective sgRNA targets were found in ITS-1 and 18S rDNA sequences of C.piscicola, respectively. In the soaking experiment, we found that the mixing of Cas9 mRNA and sgRNAs could significantly reduce the survival rate of C.piscicola. The Real-time quantitative PCR results showed that both Cas9 works with the single sgRNA or the mixture of the two sgRNA could destroy the parasite DNA at a specific region. DNA sequencing also verified the effectiveness of single sgRNA or two sgRNAs mixed with cas9 induced mutation on the target fragment of the parasite DNA in the experimental group. The results of this study once again verified the effectiveness of genetic barcoding in the application of anti-parasitic and provided a new idea for anti-parasitic methods.

Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.

Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.

Disrupting off-target Cas9 activity in the liver.

Off-target genome editing in the liver can be reduced by using lipid nanoparticles to deliver oligonucleotides that disrupt the secondary structure of single-guide RNAs as well as short interfering RNAs targeting Cas9 mRNA.

In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy.

Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.

CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles

BackgroundThe application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications.ResultsWe found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion.ConclusionsOur results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

Evaluation of CRISPR gene-editing tools in zebrafish

BackgroundZebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create ‘knockout’ models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies.MethodsTo systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression.ResultsWe compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (< 1%). To characterize if commonly used ‘mock’ CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the ‘mock’ injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation.ConclusionOverall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.

3029 – COMBINATORIAL GENE TARGETING IN PRIMARY HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

The CRISPR/Cas9 system offers enormous versatility for functional genomics. However, many applications, such as combinatorial perturbations for disease modelling and studies of gene interactions, have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, we addressed the challenge of multiplexed gene perturbations in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs) by co-delivering lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The use of fluorescent markers allows for tracing of single- or double-edited cells and we achieved robust double knockout of the cell surface markers CD45 and CD44 with an efficiency of around 70%. As a functional proof of concept, we demonstrated the use of this system to study gene dependencies for cell survival by simultaneously targeting the cohesin genes STAG1 and STAG2, as a model of synthetic lethality. Furthermore, we showed that this system can be used to study cooperative genetic events, and found a combinatorial effect with potential synergy for HSPC expansion when targeting the Aryl Hydrocarbon Receptor (AHR) in combination with members of the epigenetic modifier CoREST. Taken together, we established a paradigm for multiplexed CRISPR/Cas9 editing in primary human HSPCs, which enables studies of genetic dependencies and cooperation. This traceable multiplexed system has important implications for modelling polygenic diseases and for investigating gene interactions. The CRISPR/Cas9 system offers enormous versatility for functional genomics. However, many applications, such as combinatorial perturbations for disease modelling and studies of gene interactions, have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, we addressed the challenge of multiplexed gene perturbations in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs) by co-delivering lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The use of fluorescent markers allows for tracing of single- or double-edited cells and we achieved robust double knockout of the cell surface markers CD45 and CD44 with an efficiency of around 70%. As a functional proof of concept, we demonstrated the use of this system to study gene dependencies for cell survival by simultaneously targeting the cohesin genes STAG1 and STAG2, as a model of synthetic lethality. Furthermore, we showed that this system can be used to study cooperative genetic events, and found a combinatorial effect with potential synergy for HSPC expansion when targeting the Aryl Hydrocarbon Receptor (AHR) in combination with members of the epigenetic modifier CoREST. Taken together, we established a paradigm for multiplexed CRISPR/Cas9 editing in primary human HSPCs, which enables studies of genetic dependencies and cooperation. This traceable multiplexed system has important implications for modelling polygenic diseases and for investigating gene interactions.

Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous TP53/Trp53 genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34+ stem and progenitor cells. Strikingly, co-packaging of Cas9 mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.

Recent Progress and Future Prospective in HBV Cure by CRISPR/Cas.

Hepatitis B virus (HBV) infection remains an important issue of global public health. Although current antiviral therapy has dramatically reduced the mortality and morbidity of chronic hepatitis B (CHB), it fails to cure it. Rebound viremia often occurs after stopping antiviral therapy. Persistent HBV covalently closed circular DNA (cccDNA) and integrated DNA under antiviral therapy form the major barrier to eradication of HBV infection. CRISPR-mediated genome editing has emerged as a promising therapeutic approach to specifically destroy persistent HBV genomes, both cccDNA and integrated DNA, for HBV cure. However, the cleavage of integrated HBV DNA by CRISPR-Cas9 will cause double-strand break (DSB) of host genome, raising a serious safety concern about genome instability and carcinogenesis. The newly developed CRISPR-derived base editors (BEs), which fuse a catalytically disabled nuclease with a nucleobase deaminase enzyme, can be used to permanently inactivate HBV genome by introducing irreversible point mutations for generation of premature stop codons without DSBs of host genome. Although promising, CRISPR-mediated base editing still faces daunting challenges before its clinical application, including the base-editing efficacy, the off-target effect, the difficulty in finding conserved target HBV sequences, and in vivo delivery efficiency. Several strategies have been adopted to optimize the efficiency and specificity of CRISPR-BEs and to improve in vivo delivery efficacy through novel viral and non-viral delivery approaches. Particularly, the non-viral delivery of Cas9 mRNA and ribonucleoprotein by lipid nanoparticles exhibits attractive potential for liver-targeted delivery in clinical. Along with all progress above, the CRISPR-mediated gene therapy will ultimately achieve HBV cure.

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach.

The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology-directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient, and precise strategy for CRISPR/Cas9-mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR-amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40 bp), a synthetic single-guide RNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole genome sequencing results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders.

Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear.We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology.Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p < 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo.To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (<30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p<0.01, Figure C), blood (6-fold decrease, p<0.01), and liver (4-fold decrease, p<0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p<0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p<0.05), and IFN-γ production was reduced (p<0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E).Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. [Display omitted] DisclosuresBerrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.