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Found: 26 081
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Effective Strategies for Translating CRISPR-dCas Systems to Diverse Microbes.
CRISPR-dCas tools have widespread applications for rapidly manipulating and dissecting gene function across the microbial tree of life. However, despite their theoretical suitability for use in a broad range of species, CRISPR-dCas tools that are often initially optimized for use in model cell lines and model organisms still frequently require extensive modifications to enable their application in specific microbial organisms. Here, we review different iterations of CRISPR-dCas in microbes and the application of these techniques. We further discuss common obstacles faced and troubleshooting approaches while developing and applying CRISPR-dCas systems to a microbial organism. Finally, we suggest enhancements that can be made that may help improve the applicability of a CRISPR-dCas tool developed for nonmodel microbial organisms.
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Anti-CRISPR AcrIIC3 discriminates between Cas9 orthologs via targeting the variable surface of the HNH nuclease domain.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems constitute the adaptive immunity of bacteria and archaea, degrading nucleic acids of invading phages and plasmids. In response, phages employ anti-CRISPR (Acr) proteins as a counterdefense mechanism to neutralize the host immunity. AcrIIC3 directly inhibits target DNA cleavage of type II-C Cas9 of Neisseria meningitidis. Here, we show that AcrIIC3 interacts with the HNH nuclease domain of N. meningitidis Cas9 to inhibit its nuclease activity in an allosteric manner. The crystal structure of the AcrIIC3-HNH complex reveals that AcrIIC3 binds opposite the active site on the HNH nuclease domain. AcrIIC3 employs a unique interface for HNH, allowing it to discriminate between Cas9 orthologs, which contrasts with the broad spectrum of Cas9 inhibition by AcrIIC1. Interface residues of HNH provide key electrostatic and hydrophobic interactions that determine the host specificity of AcrIIC3. Mutations that replace HNH interfaces of N. meningitidis Cas9 with those of Geobacillus stearothermophilus Cas9 or Campylobacter jejuni Cas9 significantly attenuate AcrIIC3 binding, illustrating that the divergent interaction surface confers the host specificity of AcrIIC3. Our study demonstrates that the variable sequences of binding interface can define the target specificity of Acr proteins, suggesting potential applications in Cas9 control for gene editing.
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Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.
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Construction and Validation of a CRISPR/dCpf1-Based Transcriptional Regulatory System in Saccharomyces Cerevisiae.
Saccharomyces cerevisiae has been extensively studied and applied as a model microbial platform for efficient and sustainable production of biofuels, chemicals and natural products. In recent years, the application of CRISPR system in genome editing and transcriptional regulation has greatly improved the efficiency of microbial genome editing. To achieve transcriptional regulation the CRISPR/dCpf1-mediated CRISPRa/i transcriptional regulation system was constructed in this study. By constructing crRNA arrays and adding different activation proteins, repression efficiencies from 62% to 177% for the CRISPRi system and activation efficiencies from 154% to 320% for the CRISPRa system were achieved in the eGFP gene reporter system.
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Simultaneous Targeting of Multiple Gene Homeologs to Alter Seed Oil Production in Camelina sativa.
The ability to transform Camelina sativa easily with biosynthetic enzymes derived from other plants has made this oil seed crop an ideal platform for the production of unusual lipids valuable for different applications. However, in addition to expressing transgenic enzymes, the suppression of endogenous enzyme activity to reduce competition for common substrates or cofactors is also required to enhance the production of target compounds. As camelina possesses a relatively undifferentiated hexaploid genome, up to three gene homeologs can code for any particular enzymatic activity, complicating efforts to alter endogenous biosynthetic pathways. New genome editing technologies, such as that offered by the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system, offer the capability to introduce mutations into specifically targeted genomic sites. Here, by using a carefully designed guide RNA identical to all three homeologs, we demonstrate the ability of the CRISPR/Cas genome editing system to introduce mutations in all three CsDGAT1 or CsPDAT1 homeologous genes important for triacylglycerol (TAG) synthesis in developing seeds. Sequence analysis from transgenic T1 plants revealed that each CsDGAT1 or each CsPDAT1 homeolog was altered by multiple mutations, resulting in a genetic mosaic in the plants. Interestingly, seed harvested from both CsDGAT1- and CsPDAT1-targeted lines was often shrunken and wrinkled. Further, lipid analysis revealed that many lines produced seed with reduced oil content and altered fatty acid composition, consistent with the role of the targeted genes in seed oil biosynthesis. The CRISPR/Cas system therefore represents a useful method to alter endogenous biosynthetic pathways efficiently in polyploid species such as camelina.
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CRISPR-Cas9 technology: applications in genome engineering, development of sequence-specific antimicrobials, and future prospects.
The development of CRISPR-Cas9 technology has revolutionized our ability to edit DNA and to modulate expression levels of genes of interest, thus providing powerful tools to accelerate the precise engineering of a wide range of organisms. In addition, the CRISPR-Cas system can be harnessed to design "precision" antimicrobials that target bacterial pathogens in a DNA sequence-specific manner. This capability will enable killing of drug-resistant microbes by selectively targeting genes involved in antibiotic resistance, biofilm formation and virulence. Here, we review the origins and mechanistic basis of CRISPR-Cas systems, discuss how this technology can be leveraged to provide a range of applications in both eukaryotic and prokaryotic systems, and finish by outlining limitations and future prospects.
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CRISPR Applications in HIV Management - Prevention, Diagnosis, Monitoring and Treatment.
Despite global efforts to combat the human immunodeficiency virus (HIV) epidemic, acquired immunodeficiency syndrome (AIDS) still claims one life every minute, underscoring the persistent need for improved control strategies. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) technologies have emerged as promising tools that may transform HIV management. The objective of this review is to summarise recent advancements in CRISPR/Cas-based approaches for HIV prevention, diagnosis, monitoring, and treatment, and to evaluate their potential and current challenges. A systematic literature search was conducted to identify relevant CRISPR/Cas applications in HIV infection. In prevention, CRISPR/Cas strategies aim to hinder viral integration and enhance host immune response, although substantial development is required before clinical translation. In diagnosis, CRISPR/Cas methods show high specificity and sensitivity, yet their reliance on specialised equipment and expertise limits their accessibility. In HIV monitoring, CRISPR/Cas-based methods have not yet demonstrated superiority over the quantitative PCR. In treatment, two ongoing clinical trials - one targeting a viral co-receptor on hematopoietic stem cells (HSCs) and the other excising proviral DNA - illustrate the potential of CRISPR/Cas-mediated cures, despite challenges such as low editing efficiency and off-target effects. Overall, CRISPR/Cas technologies hold considerable promise for advancing HIV management, but issues of accessibility, affordability, and scalability must be addressed to ensure global impact.
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Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing.
Methanotrophic bacteria play a crucial role in the Earth's biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system in Methylococcus capsulatus (Bath). Heterologous coexpression of the Streptococcus pyogenes Cas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effective in vivo editing of plasmid DNA using both Cas9 and Cas9D10A nickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encoding mmoX gene with the Cas9D10A nickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing in M. capsulatus Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms.IMPORTANCE In this study, we targeted the development and evaluation of broad-host-range CRISPR/Cas9 gene-editing tools in order to enhance the genetic-engineering capabilities of an industrially relevant methanotrophic biocatalyst. The CRISPR/Cas9 system developed in this study expands the genetic tools available to define molecular mechanisms in methanotrophic bacteria and has the potential to foster advances in the generation of novel biocatalysts to produce biofuels, platform chemicals, and high-value products from natural gas- and biogas-derived methane. Further, due to the broad-host-range applicability, these genetic tools may also enable innovative approaches to overcome the barriers associated with genetically engineering diverse, industrially promising nonmodel microorganisms.
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Genome-Wide CRISPR Screening Identifies JAK1 Deficiency as a Mechanism of T-Cell Resistance.
Somatic gene mutations play a critical role in immune evasion by tumors. However, there is limited information on genes that confer immunotherapy resistance in melanoma. To answer this question, we established a whole-genome knockout B16/ovalbumin cell line by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease technology, and determined by in vivo adoptive OT-I T-cell transfer and an in vitro OT-I T-cell-killing assay that Janus kinase (JAK)1 deficiency mediates T-cell resistance via a two-step mechanism. Loss of JAK1 reduced JAK-Signal transducer and activator of transcription signaling in tumor cells-resulting in tumor resistance to the T-cell effector molecule interferon-and suppressed T-cell activation by impairing antigen presentation. These findings provide a novel method for exploring immunotherapy resistance in cancer and identify JAK1 as potential therapeutic target for melanoma treatment.
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Robust CRISPR/Cas9 mediated genome editing and its application in manipulating plant height in the first generation of hexaploid Ma bamboo (Dendrocalamus latiflorus Munro).
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