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CRISPR-Act2.0: An Improved Multiplexed System for Plant Transcriptional Activation.
CRISPR systems have greatly promoted research in genome editing and transcriptional regulation. CRISPR-based transcriptional repression and activation systems will be valuable for applications in engineering plant immunity, boosting metabolic production, and enhancing our knowledge of gene regulatory networks. Multiplexing of CRISPR allows multiple genes to be targeted without significant additional effort. Here, we describe our CRISPR-Act2.0 system which is an improved multiplexing transcriptional activation system in plants.
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Functional Evaluation of CRISPR Activity by the Dual-Fluorescent Surrogate System: C-Check.
Rapid evaluation of the CRISPR gRNA activity is an essential step of employing the technology in editing genes. Through machine learning strategy, the rule sets for in silico designing gRNAs with high activity has greatly improved. However, there are still discrepancies between different prediction rule sets, and between the predicted and actual gRNA activities. Thus, experimentally validating gRNA activity is still the gold standard in defining the best gRNAs for gene editing experiments. One such approach for experimentally selecting gRNAs with high activity is fluorescent surrogate reporter vectors. We had previously developed a dual-fluorescent surrogate system, called C-Check, which based on single-strand annealing repair of the DNA double-strand breaks introduced by CRISPR-Cas9 to generate a functional EGFP. The system offers a tool for rapid functional evaluation of CRISPR gRNA activity, as well as for enrichment of gene edited cells. In this chapter, we will give a step-by-step instruction on the design, generation, and application of the C-Check system for quantifying gRNA activities.
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High-Efficiency Genome Editing of Streptomyces Species by an Engineered CRISPR/Cas System.
Next-generation sequencing technologies have rapidly expanded the genomic information of numerous organisms and revealed a rich reservoir of natural product gene clusters from microbial genomes, especially from Streptomyces, the largest genus of known actinobacteria at present. However, genetic engineering of these bacteria is often time consuming and labor intensive, if even possible. In this chapter, we describe the design and construction of pCRISPomyces, an engineered Type II CRISPR/Cas system, for targeted multiplex gene deletions in Streptomyces lividans, Streptomyces albus, and Streptomyces viridochromogenes with editing efficiency ranging from 70% to 100%. We demonstrate pCRISPomyces as a powerful tool for genome editing in Streptomyces.
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The role of CRISPR-Cas in advancing precision periodontics.
The significant advancement of molecular biology has revolutionized medicine and provided important technologies to further clinical research development. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are DNA sequences derived from bacteriophages which have previously infected the bacterial species. The CRISPR-Cas system plays a key role in bacterial defense by detecting and destroying DNA fragments during subsequent bacteriophage invasions. The Cas9 enzyme recognizes and cleaves new invading CRISPR-complementary DNA sequences. Researchers have taken advantage of this biological device to manipulate microbes' genes and develop novel therapeutics to tackle systemic disease. In this review, we discuss the potential of utilizing CRISPR-Cas systems in the periodontal field to develop personalized periodontal care. We summarize promising attempts to bring this technology to the clinical setting. Finally, we provide insights regarding future developments to best utilize the CRISPR-Cas systems to advance precision periodontics. Although further research is imperative to evaluate the safety and potential of using CRISPR-Cas to develop precision periodontics approaches, few studies showed promising data to support the investment into this important technology in the dental sector. CRISPR-Cas9 can be a useful tool to create knockouts in vitro and in vivo as a screening tool to identify cellular pathways involved in the pathogenesis of periodontitis. Alternative CRISPR systems such as CRISPRa, CRISPRi, and Cas13 can be used to modify the transcriptome and gene expression of genes involved in periodontitis progression. CRISPR systems such as Cas3 can be used to target the periodontal biofilm and to develop new strategies to reduce or eliminate periodontal pathogens. Currently, the utility of CRISPR-Cas applications in clinical settings is limited. Through this review, we hope to foster further discussion in the periodontal research and clinical communities with respect to the potential clinical application of novel, CRISPR-Cas based, therapeutics for periodontitis.
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Visual cultures of CRISPR: intermedial figuration in science communication.
This article traces the visual culture of human genetic engineering over the past decade, focusing on the CRISPR genome editing technology. We argue that the representations surrounding CRISPR exemplify, and to an extent define, this visual culture. We examine the history of CRISPR, particularly its human applications from 2012 to 2022, through a periodization that includes the CRISPR craze, gene therapy initiatives, the He Jiankui controversy and clinical trials. Employing an expanded interpretation of intermediality within science communication, this work addresses the role of figuration across the relationships between specialist science reporting and the mainstream press and between traditional and social media. Using a mixed-methods approach combining visual and social-media analysis, the article presents an empirical analysis of three key figures - the double helix, the scientist and the human subject - and their roles across the discussed phases. The study concludes by articulating the stabilizing, amplifying and affective functions of intermedial figuration within science communication.
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The CRISPR tool kit for genome editing and beyond.
CRISPR is becoming an indispensable tool in biological research. Once known as the bacterial immune system against invading viruses, the programmable capacity of the Cas9 enzyme is now revolutionizing diverse fields of medical research, biotechnology, and agriculture. CRISPR-Cas9 is no longer just a gene-editing tool; the application areas of catalytically impaired inactive Cas9, including gene regulation, epigenetic editing, chromatin engineering, and imaging, now exceed the gene-editing functionality of WT Cas9. Here, we will present a brief history of gene-editing tools and describe the wide range of CRISPR-based genome-targeting tools. We will conclude with future directions and the broader impact of CRISPR technologies.
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Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes.
Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa). We report that Cas9d, a new CRISPR type II subtype, contains Zinc-finger motifs and high arginine content, features that we also found in nucleases related to HEARO effectors. These enzymes exhibit diverse biochemical characteristics and are broadly targetable. We show that natural Cas9d enzymes are capable of genome editing in mammalian cells with >90% efficiency, and further engineered nickase variants into the smallest base editors active in E. coli and human cells. Their small size, broad targeting potential, and translatability suggest that Cas9d and HEARO systems will enable a variety of genome editing applications.
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Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives.
According to Darwin's theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous areas of life, and CRISPR-Cas represents the most suitable strategy. The system has ignited a revolution in the field of genetic engineering. The ease, precision, affordability of this system is akin to a Midas touch for researchers editing genomes. Undoubtedly, the applications of this system are endless. The CRISPR-Cas system is extensively employed in the treatment of infectious and genetic diseases, in metabolic disorders, in curing cancer, in developing sustainable methods for fuel production and chemicals, in improving the quality and quantity of food crops, and thus in catering to global food demands. Future applications of CRISPR-Cas will provide benefits for everyone and will save countless lives. The technology is evolving rapidly; therefore, an overview of continuous improvement is important. In this review, we aim to elucidate the current state of the CRISPR-Cas revolution in a tailor-made format from its discovery to exciting breakthroughs at the application level and further upcoming trends related to opportunities and challenges including ethical concerns.
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The CRISPR-Cas toolbox and gene editing technologies.
The emergence of CRISPR-Cas systems has accelerated the development of gene editing technologies, which are widely used in the life sciences. To improve the performance of these systems, workers have engineered and developed a variety of CRISPR-Cas tools with a broader range of targets, higher efficiency and specificity, and greater precision. Moreover, CRISPR-Cas-related technologies have also been expanded beyond making cuts in DNA by introducing functional elements that permit precise gene modification, control gene expression, make epigenetic changes, and so on. In this review, we introduce and summarize the characteristics and applications of different types of CRISPR-Cas tools. We discuss certain limitations of current approaches and future prospects for optimizing CRISPR-Cas systems.
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Cas3 Protein-A Review of a Multi-Tasking Machine.
Cas3 has essential functions in CRISPR immunity but its other activities and roles, in vitro and in cells, are less widely known. We offer a concise review of the latest understanding and questions arising from studies of Cas3 mechanism during CRISPR immunity, and highlight recent attempts at using Cas3 for genetic editing. We then spotlight involvement of Cas3 in other aspects of cell biology, for which understanding is lacking-these focus on CRISPR systems as regulators of cellular processes in addition to defense against mobile genetic elements.
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