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Engineered virus-like particles for in vivo gene editing ameliorate hearing loss in murine DFNA2 model.
Although gene editing therapy is applicable to human diseases, its efficiency and safety require further investigation. Further, non-virus-mediated gene editor delivery is challenging in the inner ear. Here, engineered virus-like particles (eVLPs) were used for inner ear delivery of SpCas9 and single-guided RNA to delete the Kcnq4 dominant-negative mutant allele, which causes progressive hearing loss in a non-syndromic hearing loss murine model. eVLP-delivered SpCas9 was administered to the inner ears of Kcnq4W277S/+ mice to target the Kcnq4-expressing outer hair cells (OHCs). Hearing loss was significantly alleviated 7 weeks after eVLP administration. OHC survival improved significantly, and OHC-innervating neurite (connected to type II spiral ganglion neuronal body) loss was ameliorated. Finally, OHC membrane potential was hyperpolarized with eVLP gene editor treatment in Kcnq4-mutant mice, indicating that their OHCs were healthier and more stable than those of uninjected mice. Our findings suggest that eVLPs are feasible inner ear gene editor deliverers to treat hearing loss.
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CRISPR/Cas9-Mediated Genome Editing in Zebrafish.
The CRISPR/Cas9 system is a powerful tool for genome editing in zebrafish. This workflow takes advantage of the genetic tractability of zebrafish and will allow users to edit genomic sites and produce mutant lines using selective breeding. Established lines may then be employed by researchers for downstream genetic and phenotypic analyses.
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Efficiency of genetic modification in gene-knockout sperm-derived zygotes followed by electroporation of guide RNA targeting the same gene.
Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene-knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild-type and gene-deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene-deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification.
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Further analysis of barley MORC1 using a highly efficient RNA-guided Cas9 gene-editing system.
Microrchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL-type ATPase and an S5-fold. In plants, MORC proteins were first discovered in a genetic screen for Arabidopsis thaliana mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and genome stabilization. Little is known about the role of MORC proteins of cereals, especially because knockout (KO) mutants were not available and assessment of loss of function relied only on RNAi strategies, which were arguable, given that MORC proteins in itself are influencing gene silencing. Here, we used a Streptococcus pyogenes Cas9 (SpCas9)-mediated KO strategy to functionally study HvMORC1, one of the current seven MORC members of barley. Using a novel barley RNA Pol III-dependent U3 small nuclear RNA (snRNA) promoter to drive expression of the synthetic single guide RNA (sgRNA), we achieved a very high mutation frequency in HvMORC1. High frequencies of mutations were detectable by target sequencing in the callus, the T0 generation (77%) and T1 generation (70%-100%), which constitutes an important improvement of the gene-editing technology in cereals. Corroborating and extending earlier findings, SpCas9-edited hvmorc1-KO barley, in clear contrast to Arabidopsis atmorc1 mutants, had a distinct phenotype of increased disease resistance to fungal pathogens, while morc1 mutants of either plant showed de-repressed expression of transposable elements (TEs), substantiating that plant MORC proteins contribute to genome stabilization in monocotyledonous and dicotyledonous plants.
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gEL DNA: A Cloning- and Polymerase Chain Reaction-Free Method for CRISPR-Based Multiplexed Genome Editing.
Even for the genetically accessible yeast Saccharomyces cerevisiae, the CRISPR-Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events, and diverse approaches for multiplex genome editing have been described in the literature by means of SpCas9 or FnCas12a endonucleases and their associated guide RNAs (gRNAs). The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native Pol II or Pol III RNA polymerases. These gRNA expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in Saccharomyces cerevisiae. Using FnCas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single and duplex DNA editing. These DNA fragments can be custom ordered, which makes this approach highly suitable for high-throughput strain construction. This work expands the CRISPR toolbox for large-scale strain construction programs in S. cerevisiae and promises to be relevant for other less genetically accessible yeast species.
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[Gene editing in drug discovery and therapeutic innovation].
The idea according to which the most recent therapeutic methods will overcome the more traditional pharmacopoeia is widespread in recent publications. Biomedicine and gene therapies are booming, but we realize, as for other therapeutic approaches, that they suffer intrinsic constraints and limitations and that their most relevant therapeutic fields are complementary to those of traditional drugs. They are now viewed as potentially synergistic with these traditional drugs, rather than competitors. This review puts into perspective the potential of genome editing in the field of drug discovery and therapeutic innovation.
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Learning from the Past: Discussing Lessons from Reproductive Justice in the Gene-Editing Sphere.
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CRISPR-Cas9 as a Trojan horse.
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Genome Editing, Transcriptional Regulation, and Forward Genetic Screening Using CRISPR-Cas12a Systems in Yarrowia lipolytica.
Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host.
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Integrate CRISPR/Cas9 for protein expression of HLA-B*38:68Q via precise gene editing.
The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. We studied the expression level of a questionable (Q) HLA-B*38:68Q allele, which carries a 9-nucleotide (nt) deletion at codon 230-232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 gene editing of HLA-B*38:01:01:01 homozygous EBV B cell line resulted in one HLA-B*38:68Q/B*38:01:01:01 heterozygous and one HLA-B*38:68Q homozygous clone. Flow cytometric analysis of monoclonal anti-Bw4 antibody showed the protein expression of HLA-B*38:01:01:01 in homozygous cells was 2.2 fold higher than HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the expression of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 2.0 fold higher than HLA-B*38:68Q homozygous cells. The HLA-B*38:68Q expression was further confirmed using anti-B38 polyclonal antibody. Similarly, the expression of the HLA-B*38:01:01:01 homozygous cells was 1.5 fold higher than that of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 1.6 fold higher than that of HLA-B*38:68Q homozygous cells. The treatment of HLA-B*38:68Q homozygous cells with IFN-γ significantly increased its expression. In conclusion, we demonstrate that HLA-B*38:68Q is a low-expressing HLA allele. The CRISPR/Cas9 technology is a useful tool to induce precise gene editing in HLA genes to enable the characterization of HLA gene variants on expression and function.
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