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CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes.
CRISPR-Cas9-mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are no reptiles with effective methods for targeted mutagenesis. Here, we demonstrate that the microinjection of immature oocytes within the ovaries of Anolis sagrei females enables the production of CRISPR-Cas9-induced mutations. This method is capable of producing F0 embryos and hatchlings with monoallelic or biallelic mutations. We demonstrate that these mutations can be transmitted through the germline to establish genetically modified strains of lizards. Direct tests of gene function can now be performed in Anolis lizards, an important model for studies of reptile evolution and development.
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Establishment of a Secretory Protein-Inducible CRISPR/Cas9 System for Nosema bombycis in Insect Cells.
Gene editing techniques are widely and effectively used for the control of pathogens, but it is difficult to directly edit the genes of Microsporidia due to its unique spore wall structure. Innovative technologies and methods are urgently needed to break through this limitation of microsporidia therapies. Here, we establish a microsporidia-inducible gene editing system through core components of microsporidia secreted proteins, which could edit target genes after infection with microsporidia. We identified that Nosema bombycis NB29 is a secretory protein and found to interact with itself. The NB29-N3, which lacked the nuclear localization signal, was localized in the cytoplasm, and could be tracked into the nucleus after interacting with NB29-B. Furthermore, the gene editing system was constructed with the Cas9 protein expressed in fusion with the NB29-N3. The system could edit the exogenous gene EGFP and the endogenous gene BmRpn3 after overexpression of NB29 or infection with N. bombycis.
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Treating genetic blood disorders in the era of CRISPR-mediated genome editing.
In the setting of monogenic disease, advances made in genome editing technologies can, in principle, be deployed as a therapeutic strategy to precisely correct a specific gene mutation in an affected cell type and restore functionality. Using the β-hemoglobinopathies and hemophilia as exemplars, we review recent experimental breakthroughs using CRISPR-derived genome editing technology that have translated to significant improvements in the management of inherited hematologic disorders. Yet there are also challenges facing the use of CRISPR-mediated genome editing in these patients; we discuss possible ways to obviate those issues for furtherance of clinical benefit.
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Genome editing in hymenoptera.
The application of genome editing tools in Hymenoptera has transformative potential for functional genetics and understanding their unique biology. Hymenoptera comprise one of the most diverse Orders of animals, and the development of methods for efficiently creating precise genome modifications could have applications in conservation, pest management and agriculture. To date, sex determination, DNA methylation, taste and smell sensory systems as well as phenotypic markers have been selected for gene editing investigations. From these data, insights into eusociality, the nature of haplodiploidy and the complex communication systems that Hymenoptera possess have provided an understanding of their evolutionary history that has led them to become so diverse and successful. Insights from these functional genetics analyses have been supported by the ever-improving suite of CRIPSR tools and further expansion will allow more specific biological hypotheses to be tested and applications beyond the lab. Looking ahead, genome editing tools have potential for Hymenopteran applications in modifying biocontrol agents of agricultural pests and for use in managing invasive species through the development of technologies such as gene drives. This review provides accessibility to information regarding the status of Hymenopteran genome editing, intending to support the considered development of CRISPR tools in novel species as well as innovation and refinement of methods in species in which it has already been achieved.
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Kazuto Kato: the ethics of editing humanity.
Gary Humphreys talks to Kazuto Kato about the ethical and societal challenges posed by biotechnologies that allow for the editing of the human genome.
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Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications.
The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes.
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Prime editing in chicken fibroblasts and primordial germ cells.
CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. However, there has been no report of taking advantage of the PE system for gene editing in primordial germ cells (PGCs) thus far. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and PGCs. By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chicken fibroblasts and PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.
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Design and Validation of Guide RNAs for CRISPR-Cas9 Genome Editing in Mosquitoes.
CRISPR-Cas9 has revolutionized gene editing for traditional and nontraditional model organisms alike. This tool has opened the door to new mechanistic studies of basic mosquito biology as well as the development of novel vector control strategies based on CRISPR-Cas9, including gene drives that spread genetic elements in the population. Although the promise of the specificity, flexibility, and ease of deployment CRISPR is real, its implementation still requires empirical optimization for each new species of interest, as well as to each genomic target within a given species. Here, we provide an overview of designing and testing single-guide RNAs for the use of CRISPR-based gene editing tools.
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CRISPR-Editing Therapy for Duchenne Muscular Dystrophy.
Duchenne muscular dystrophy (DMD) is a debilitating genetic disorder that results in progressive muscle degeneration and premature death. DMD is caused by mutations in the gene encoding dystrophin protein, a membrane-associated protein required for maintenance of muscle structure and function. Although the genetic mutations causing the disease are well known, no curative therapies have been developed to date. The advent of genome-editing technologies provides new opportunities to correct the underlying mutations responsible for DMD. These mutations have been successfully corrected in human cells, mice, and large animal models through different strategies based on CRISPR-Cas9 gene editing. Ideally, CRISPR-editing could offer a one-time treatment for DMD by correcting the genetic mutations and enabling normal expression of the repaired gene. However, numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene-editing components to all the muscles of the body, and the suppression of possible immune responses to the CRISPR-editing therapy. This review provides an overview of the recent advances toward CRISPR-editing therapy for DMD and discusses the opportunities and the remaining challenges in the path to clinical translation.
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CRISPR/Cas9-mediated genome editing in sea urchins.
The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9) technology enables rapid, targeted, and efficient changes in the genomes of various model organisms. The short guide RNAs (gRNAs) of the CRISPR/Cas9 system can be designed to recognize target DNA within coding regions for functional gene knockouts. Several studies have demonstrated that the CRISPR/Cas9 system efficiently and specifically targets sea urchin genes and results in expected mutant phenotypes. In addition to disrupting gene functions, modifications and additions to the Cas9 protein enable alternative activities targeted to specific sites within the genome. This includes a fusion of cytidine deaminase to Cas9 (Cas9-DA) for single nucleotide conversion in targeted sites. In this chapter, we describe detailed methods for the CRISPR/Cas9 application in sea urchin embryos, including gRNA design, in vitro synthesis of single guide RNA (sgRNA), and the usages of the CRISPR/Cas9 technology for gene knockout and single nucleotide editing. Methods for genotyping the resultant embryos are also provided for assessing efficiencies of gene editing.
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