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The clinical cutting edge.
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CRISPR-Mediated Genome Editing in the Human Fungal Pathogen C. albicans.
Cas9-mediated genome editing is one tool investigators can use to study fungal pathogens. Such methodologies allow the investigator to examine how fungal cells differ from human cells and thus potentially identify novel therapeutic targets. In this chapter, we describe how CRISPR-mediated genome editing can be used to edit the genome of the most prevalent human fungal pathogen C. albicans. A cassette encoding a fungal optimized Cas9 nuclease and guide RNA is integrated into the C. albicans genome. The guide RNA targets Cas9 to the complementary genome sequence, and Cas9 cleaves the DNA. A repair template encoding whatever changes the investigator wished to make to the genome is co-transformed with the cassette and repairs the break via homologous recombination, thus introducing the change to the genome. The method we describe enables the researcher to edit the C. albicans genome and then efficiently remove the editing machinery and antibiotic resistance markers. This allows one to sequentially edit the C. albicans genome when multiple changes are desired. In addition, we provide notes that provide guidance on how the described protocols can be altered to meet the demands of the researcher. In these notes, we also describe the recent development of a more flexible CRISPR system that has a relaxed PAM site specificity. These and other advancements make CRISPR-mediated genome editing a practical approach when one needs to genetically alter C. albicans.
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Immortalised Cas9-expressing Cell lines for Gene interrogation.
The ability of modifying the genome of multiple species, precisely and without or minimal off-targeted effects, have opened numerous opportunities for the biotechnology industry. In this chapter, we describe an easy to establish, robust, and practical pipeline that can be used to generate immortalized cell lines, from different tissues, to capture cell linage context and validate the tools required for genome editing and genetic modification. This pipeline serves as a reference for similar approaches for gene interrogation in other species.
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The Human Genome Editing Race: Loosening Regulatory Standards for Commercial Advantage?
Medicinal products based on genome editing must undergo rigorous preclinical testing and are subject to regulatory oversight for proper risk assessment prior to first evaluation in humans. We give a European perspective on the regulatory expectations to translate genome editing to the clinic to ensure their timely progress to market.
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Improvement of Rice Agronomic Traits by Editing Type-B Response Regulators.
Type-B response regulator proteins in rice contain a conserved receiver domain, followed by a GARP DNA binding domain and a longer C-terminus. Some type-B response regulators such as RR21, RR22 and RR23 are involved in the development of rice leaf, root, flower and trichome. In this study, to evaluate the application potential of type-B response regulators in rice genetic improvement, thirteen type-B response regulator genes in rice were respectively knocked out by using CRISPR/Cas9 genome editing technology. Two guide RNAs (gRNAs) were simultaneously expressed on a knockout vector to mutate one gene. T0 transformed plants were used to screen the plants with deletion of large DNA fragments through PCR with specific primers. The mutants of CRISPR/Cas9 gene editing were detected by Cas9 specific primer in the T1 generation, and homozygous mutants without Cas9 were screened, whose target regions were confirmed by sequencing. Mutant materials of 12 OsRRs were obtained, except for RR24. Preliminary phenotypic observation revealed variations of various important traits in different mutant materials, including plant height, tiller number, tillering angle, heading date, panicle length and yield. The osrr30 mutant in the T2 generation was then further examined. As a result, the heading date of the osrr30 mutant was delayed by about 18 d, while the yield was increased by about 30%, and the chalkiness was significantly reduced compared with those of the wild-type under field high temperature stress. These results indicated that osrr30 has great application value in rice breeding. Our findings suggest that it is feasible to perform genetic improvement of rice by editing the type-B response regulators.
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Walking a Fine Germline: Synthesizing Public Opinion and Legal Precedent to Develop Policy Recommendations for Heritable Gene-Editing.
Gene-editing technologies, such as CRISPR/Cas9, are internationally ethically fraught. In the United States, policy surrounding gene-editing has yet to be implemented, while the science continues to speed ahead. However, it is not enough that policy be implemented: in order for policy to establish limits for the technology such that benefits are possible while threats are kept at bay, such policy must be ethical. In turn, the ethics of gene-editing is a culturally determined field of inquiry. This piece presents a proposal for a study whose goal is to arrive at ethical policy recommendations for policymakers. To achieve this goal, this study proposes, what needs to be done is, first, to understand the full history and foundation of gene-editing by conducting a thorough legal, bioethical, and policy review for precedent assisted reproductive technologies and genetic reproductive technologies. Following this effort, an empirical study must be conducted involving careful surveys of key stakeholder groups on their knowledge and opinions of gene-editing. Such stakeholder groups must include bioethicists, medical geneticists, and lay persons, including those in the disability community.
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Transgene-Free Genome Editing in Nicotiana benthamiana with CRISPR/Cas9 Delivered by a Rhabdovirus Vector.
The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas systems have become the most widely adopted genome editing platform owing to their unprecedented simplicity, efficiency, and versatility. Typically, the genome editing enzyme is expressed in plant cells from an integrated transgene delivered by either Agrobacterium-mediated or biolistic transformation. Recently, plant virus vectors have emerged as promising tools for the in planta delivery of CRISPR/Cas reagent. Here, we provide a protocol for CRISPR/Cas9-mediated genome editing in the model tobacco plant Nicotiana benthamiana using a recombinant negative-stranded RNA rhabdovirus vector. The method is based on infection of N. benthamiana with a Sonchus yellow net virus (SYNV)-based vector that carries the Cas9 and guide RNA expression cassettes to target specific genome loci for mutagenesis. With this method, mutant plants free of foreign DNA can be obtained within 4-5 months.
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Simple CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae.
CRISPR-Cas9 has emerged as a powerful method for editing the genome in a wide variety of species, since it can generate a specific DNA break when targeted by the Cas9-bound guide RNA. In yeast, Cas9-targeted DNA breaks are used to promote homologous recombination with a mutagenic template DNA, in order to rapidly generate genome edits (e.g., DNA substitutions, insertions, or deletions) encoded in the template DNA. Since repeated Cas9-induced DNA breaks select against unedited cells, Cas9 can be used to generate marker-free genome edits. Here, we describe a simple protocol for constructing Cas9-expressing plasmids containing a user-designed guide RNA, as well as protocols for using these plasmids for efficient genome editing in yeast. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Constructing the guide RNA expression vector Basic Protocol 2: Preparing double-stranded oligonucleotide repair template Alternate Protocol 1: Preparing a single-stranded oligonucleotide repair template Basic Protocol 3: Induce genome editing by co-transformation of yeast Basic Protocol 4: Screening for edited cells Basic Protocol 5: Removing sgRNA/CAS9 expression vector Alternate Protocol 2: Removing pML107-derived sgRNA/CAS9 expression vector.
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What if Bacteria Could Tell Us What They Have Seen?
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RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production.
Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.
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