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Radical Technology Meets Radical Application: An Interview with George Church.
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Fluorescently Labeled TracrRNA Improves Work Flow and Facilitates Successful Genome Editing in Zebrafish.
Gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) is widely used throughout the zebrafish community for the generation of knockouts and knockins. One of the bottlenecks that exists during the process is the laborious screening of injected embryos for F0 founder fish or CRISPants, weeks after the injection date. In this study we show that the use of fluorescently tagged tracrRNA and sorting for fluorescent embryos as early as the 512-cell stage using stereomicroscope significantly improve yield of fish with successfully CRISPR/Cas9-edited genomes. This is a cost-effective strategy that significantly improves workflow and efficacy in genome editing in particular for less experienced researchers.
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Clustered Regularly Interspaced Short Palindromic Repeats System of Genome Engineering in Embryos to Repair Genes.
CRISPR is a powerful gene editing tool for correcting disease-causing mutations. It is becoming more and more evident that CRISPR is a promising approach to treating human genetic diseases. The technologies for adding or removing genes have made significant advances over the past few years and have shown promising potential outcomes. In the current study, we mainly introduce the CRISPR/Cas system and there are several applications in the treatment of genetic diseases, particularly during embryo development.
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Gene edits to 'CRISPR babies' might have shortened their life expectancy.
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Generation of gene-edited sheep with a defined Booroola fecundity gene (FecBB) mutation in bone morphogenetic protein receptor type 1B (BMPR1B) via clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9.
Since its emergence, the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 system has been increasingly used to generate animals for economically important traits. However, most CRISPR/Cas9 applications have been focused on non-homologous end joining, which results in base deletions and insertions, leading to a functional knockout of the targeted gene. The Booroola fecundity gene (FecBB) mutation (p.Q249R) in bone morphogenetic protein receptor type 1B (BMPR1B) has been demonstrated to exert a profound effect on fecundity in many breeds of sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.249Q>R substitution through the coinjection of Cas9 mRNA, a single guide RNA and single-stranded DNA oligonucleotides into Tan sheep zygotes. In the newborn lambs, the observed efficiency of the single nucleotide exchange was as high as 23.8%. We believe that our findings will contribute to improved reproduction traits in sheep, as well as to the generation of defined point mutations in other large animals.
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Upstream Ethical Mapping of Germline Genome Editing.
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Broadening the applicability of CRISPR/Cas9 in plants.
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Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an efficient method for the production of gene-edited animals. We have successfully generated gene-modified goats and sheep via zygote injection of Cas9 mRNA and single-guide RNA (sgRNA) mixtures. However, the delivery system for microinjection largely refers to methods established for mice; optimised injection conditions are urgently required for the generation of large animals. Here, we designed a study to optimise the Cas9 mRNA and sgRNA delivery system for goats. By comparing four computational tools for sgRNA design and validating the targeting efficiency in goat fibroblasts, we suggest a protocol for the selection of desirable sgRNAs with higher targeting efficiency and negligible off-target mutations. We further evaluated the editing efficiency in goat zygotes injected with Cas9:sgRNA (sg8) and found that injection with 50ngμL-1 Cas9 mRNA and 25ngμL-1 sgRNA yielded an increased editing efficiency. Our results provide a reference protocol for the optimisation of the injection conditions for the efficient editing of large animal genomes via the zygote injection approach.
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Cutting Through the "Gray Area": An Analysis of the IBC Regulatory Oversight of Applications of CRISPR Technology in Clinical Research.
The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technology has quickly ushered in a new era of gene editing and offered exciting potential for the development of biomedical products. While the breadth of applications for CRISPR encompasses nearly the entire field of medical science, its utilization to produce next-generation CAR T cells stands to benefit most substantially in the short-term. These novel therapeutics are now beginning to enter the clinical trial phase of the numerous approval pipelines, but does the existing regulatory framework have the capability to adequately review and approve the initiation of these new age research endeavors in the clinical setting? This analysis will illuminate the similarity and differences between CRISPR developed interventions and existing techniques used to produce biomedical products from a regulatory perspective in the United States. Further, the "gray area" surrounding CRISPR regulatory oversight will be explored and recommendations will be made to facilitate the elimination of inconsistencies that currently exist in the assessment of this work prior to the initiation of associated clinical trials.
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CRISPR patent ruling leaves license holders scrambling.
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