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Tweaking genes with CRISPR or viruses fixes blood disorders.
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Naïve Primary Mouse CD8+ T Cells Retain In Vivo Immune Responsiveness After Electroporation-Based CRISPR/Cas9 Genetic Engineering.
CRISPR/Cas9 technology has revolutionized genetic engineering of primary cells. Although its use is gaining momentum in studies on CD8+ T cell biology, it remains elusive to what extent CRISPR/Cas9 affects in vivo function of CD8+ T cells. Here, we optimized nucleofection-based CRISPR/Cas9 genetic engineering of naïve and in vitro-activated primary mouse CD8+ T cells and tested their in vivo immune responses. Nucleofection of naïve CD8+ T cells preserved their in vivo antiviral immune responsiveness to an extent that is indistinguishable from non-nucleofected cells, whereas nucleofection of in vitro-activated CD8+ T cells led to slightly impaired expansion/survival at early time point after adoptive transfer and more pronounced contraction. Of note, different target proteins displayed distinct decay rates after gene editing. This is in stark contrast to a comparable period of time required to complete gene inactivation. Thus, for optimal experimental design, it is crucial to determine the kinetics of the loss of target gene product to adapt incubation period after gene editing. In sum, nucleofection-based CRISPR/Cas9 genome editing achieves efficient and rapid generation of mutant CD8+ T cells without imposing detrimental constraints on their in vivo functions.
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Self-inactivating AAV-CRISPR at different ages enables sustained amelioration of Huntington's disease deficits in BAC226Q mice.
Huntington's disease (HD) is a monogenic autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HTT gene, yielding a gain-of-toxic-function mutant Huntingtin protein (mHTT). CRISPR-Cas9 is a potentially powerful therapeutic strategy for HD by eliminating mutant HTT (mHTT) gene. We developed a specific SaCas9 guide RNA to target human mHTT and a self-inactivating gene editing system that abolishes SaCas9 after a short transient expression for high gene editing efficiency and maximal safety to prevent off-target effects. Both conventional and the self-inactivating gene editing systems successfully eliminated mHTT gene, 60 to 90% mHTT protein and 90% of mHTT aggregation in BAC226Q mouse brains, which resulted in significant long-term rescue of neuropathology, motor deficits, weight loss, and shortened life span. These beneficial effects were observed when gene editing was applied before, at, and well after the onset of pathological and behavioral abnormalities. These proof-of-concept data demonstrate that gene editing can be a highly effective therapeutic approach for HD.
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CRISPR-Cas9 as a Trojan horse.
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Protocol for CRISPR-Cas9 genome editing of a swine cell line via electroporation.
Genome editing technology is being used in animals for a variety of purposes, including improvement of animal and public health outcomes. Characterization of genome editing reagents and anticipated genomic alterations is an essential step toward the development of an edited animal. Here, we present a protocol for genome editing in the swine testicular (ST) cell line. We describe steps for evaluating CRISPR-Cas9 complex functionality in vitro, delivering editing molecules into cells by transfection, and assessing target editing via Sanger sequencing.
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Simple, Efficient CRISPR-Cas9-Mediated Gene Editing in Mice: Strategies and Methods.
Genetic modification of almost any species is now possible using approaches based on targeted nucleases. These novel tools now bypass previous limited species windows, allowing precision nucleotide modification of the genome at high efficiency, rapidly and economically. Here we focus on the modification of the mouse genome; the mouse, with its short generation time and comparatively low maintenance/production costs is the perfect mammal with which to probe the genome to understand its functions and complexities. Further, using targeted nucleases combined with homologous recombination, it is now possible to precisely tailor the genome, creating models of human diseases and conditions directly and efficiently in zygotes derived from any mouse strain. Combined these approaches make it possible to sequentially and progressively refine mouse models to better reflect human disease, test and develop therapeutics. Here, we briefly review the strategies involved in designing targeted nucleases (sgRNAs) providing solutions and outlining in detail the practical processes involved in precision targeting and modification of the mouse genome and the establishing of new precision genetically modified mouse lines.
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Falling giants and the rise of gene editing: ethics, private interests and the public good.
This paper considers the tensions created in genomic research by public and private for-profit ideals. Our intent is to strengthen the public good at a time when doing science is strongly motivated by market possibilities and opportunities. Focusing on the emergence of gene editing, and in particular CRISPR, we consider how commercialisation encourages hype and hope-a sense that only promise and idealism can achieve progress. At this rate, genomic research reinforces structures that promote, above all else, private interests, but that may attenuate conditions for the public good of science. In the first part, we situate genomics using the aphorism that 'on the shoulders of giants we see farther'; these giants are infrastructures and research cultures rather than individual 'heroes' of science. In this respect, private initiatives are not the only pivot for successful discovery, and indeed, fascination in those could impinge upon the fundamental role of public-supported discovery. To redress these circumstances, we define the extent to which progress presupposes research strategies that are for the public good. In the second part, we use a 'falling giant' narrative to illustrate the risks of over-indulging for-profit initiatives. We therefore offer a counterpoint to commercialised science, using three identifiable 'giants'-scientists, publics and cultures-to illustrate how the public good contributes to genomic discovery.
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Transcriptomics and phenotypic analysis of OTOF gene knockdown in zebrafish mediated by CRISPR/Cas9.
Deafness is a common genetic disorder, where mutations,in the OTOF gene can disrupt the normal functionof the Otoferlin protein, leading to impaired neurotransmitter release in the inner ear and subsequent deafness. Despite the complexity of the pathogenic mechanism,it is not fully understood. Zebrafish are an excellent model for studying genetically-induced deafness,but there have been no previous reports on the pathogenesis of OTOF in zebrafish.This study successfully established a zebrafish model with mutated OTOF genes using CRISPR/Cas9 gene editing technology to investigate the molecular basis of OTOF-induced deafness. Compared to AB wild type zebrafish, those with low otof expression showed injury and apoptosis of hair cells in the posterior lateral neuromasts along with significant increase in the number of macrophages and apoptotic cells in this region. Additionally, these mutants exhibited a reduction in body length. To further elucidate differences at 5dpf (days post-fertilization) between mutant and wild type zebrafish embryos, RNA-seq analysis was conducted to examine differentially expressed genes (DEGs).A total of 334 up-regulated DEGs and 111 down-regulated DEGs were identified in mutants compared to wild types.KEGG and GO enrichment analyses were performed on these DEGs to identify key signaling pathways and hub DEGs. The findings revealedan increased expression of several genes involved in the HSP70 oxidative stress system, suggesting that OTOF may protect cochlear hair cell from apoptosis induced by oxidative stress through regulation of MAPK signal and HSP70 expression.In summary, the establishment of a zebrafish model with OTOF knockout provides a valuable tool for investigating the function of Otoferlin and understanding the role of the OTOF gene in deafness. These potential molecular insights offer significant contributions towards understanding the pathogenesis of deafness experimental models and serves as a foundation for comprehending the involvement of the OTOF gene.
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Gene editing of the thioester reductase step in the biosynthesis of lysergic acid amides.
Ergot alkaloids derived from lysergic acid are important in agriculture, as food and feed contaminants, and in medicine, as the foundation of several pharmaceuticals. The fungus Metarhizium brunneum makes several lysergic acid amides, with lysergic acid α-hydroxyethylamide (LAH) being produced in by far the highest concentration. The multifunctional enzyme lysergyl peptide synthetase 3 (Lps3) has multiple domains that play important roles in lysergic acid amide synthesis. We hypothesized a role for the reductase domain of Lps3 in liberating LAH from an enzyme-bound precursor and tested this hypothesis with CRISPR/Cas9-based gene editing experiments. We transformed M. brunneum with a Cas9/single guide RNA complex and a donor DNA that replaced the tyrosine at the active site of the reductase domain of Lps3 with a phenylalanine. Sanger sequencing of edited and wild-type genes demonstrated successful editing of the reductase domain without non-target mutations in Lps3. High performance liquid chromatography of the edited strain showed a significant reduction of LAH and accumulation of the precursor lysergic acid. The phenotype was similar when the edited allele of lpsC was in a wild-type background or in backgrounds with late pathway genes easO or easP knocked out, except no LAH was detectable when the edit was in the easO knockout background. The data demonstrate that the reductase domain plays a key role or roles in formation of LAH. The abundant lysergic acid accumulating in the mutants, as opposed to later pathway intermediates in LAH biosynthesis (such as lysergyl-alanine), indicated severe debilitation of Lps3. The data indicate a requirement for the reductase domain of Lps3 in synthesis of lysergic acid amides and demonstrate the feasibility of the CRISPR/Cas9-based approach for editing genes in Metarhizium species.
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Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing.
In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al.1.
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