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Current and Prospective Applications of CRISPR-Cas12a in Pluricellular Organisms.
CRISPR-Cas systems play a critical role in the prokaryotic adaptive immunity against mobile genetic elements, such as phages and foreign plasmids. In the last decade, Cas9 has been established as a powerful and versatile gene editing tool. In its wake, the novel RNA-guided endonuclease system CRISPR-Cas12a is transforming biological research due to its unique properties, such as its high specificity or its ability to target T-rich motifs, to induce staggered double-strand breaks and to process RNA arrays. Meanwhile, there is an increasing need for efficient and safe gene activation, repression or editing in pluricellular organisms for crop improvement, gene therapy, research model development, and other goals. In this article, we review CRISPR-Cas12a applications in pluricellular organisms and discuss how the challenges characteristic of these complex models, such as vectorization or temperature variations in ectothermic species, can be overcome.
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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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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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Advances in and Perspectives on Transgenic Technology and CRISPR-Cas9 Gene Editing in Broccoli.
Broccoli, a popular international Brassica oleracea crop, is an important export vegetable in China. Broccoli is not only rich in protein, vitamins, and minerals but also has anticancer and antiviral activities. Recently, an Agrobacterium-mediated transformation system has been established and optimized in broccoli, and transgenic transformation and CRISPR-Cas9 gene editing techniques have been applied to improve broccoli quality, postharvest shelf life, glucoraphanin accumulation, and disease and stress resistance, among other factors. The construction and application of genetic transformation technology systems have led to rapid development in broccoli worldwide, which is also good for functional gene identification of some potential traits in broccoli. This review comprehensively summarizes the progress in transgenic technology and CRISPR-Cas9 gene editing for broccoli over the past four decades. Moreover, it explores the potential for future integration of digital and smart technologies into genetic transformation processes, thus demonstrating the promise of even more sophisticated and targeted crop improvements. As the field continues to evolve, these innovations are expected to play a pivotal role in the sustainable production of broccoli and the enhancement of its nutritional and health benefits.
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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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Glucocorticoid receptor quaternary structure drives chromatin occupancy and transcriptional outcome.
Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor NR3C1 (also known as GR) forms tetramers on enhancers, owing to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. By using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knockout cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to the wild-type receptor and both induces and represses more genes than GRwt. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher-order structure that drives both the activating and repressive actions of glucocorticoids.
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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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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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Miniaturized CRISPR: Ultra Compact Systems for In Vivo Delivery and Portable Diagnostics.
Reduced-size CRISPR systems have become a possible remedy to the delivery and size constraints of the traditional SpCas9 (~ 1368 Å). Recently described small nucleases, including Cas12f (400-700 Å) or CasX (~ 980 Å), along with designed mini-Cas9 versions, can efficiently be used in vivo to edit cells as well as to perform point-of-care diagnostics because of their lower molecular weight and less complex structures. This review will sum up progress in compact Cas protein engineering, guide RNA optimization, and delivery vector miniaturization, and point to their influence in therapeutic gene editing and portable diagnostic platforms. We additionally cover the contemporary issues of interest, such as off-target activity, delivery barriers and regulatory requirements, and future opportunities provided through AI-assisted protein design and synthetic biology. The miniaturized CRISPR technology is bound to substantially transform the translational arena of gene editing and world diagnostics.
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