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[Gene editing technology and its research progress in China].
Gene editing is a technology for precision modification of genome sequences or expressing transcripts. Early versions of such techniques include homing endonuclease, zinc finger endonuclease and transcription-like activator effectors. The CRISPR/Cas9 system has recently emerged as a powerful and versatile gene editing tool for a broad spectrum of applications. In this review, we discuss the recent development and applications of gene editing technology, and particularly focus on the progress in China, with the emphasis on mechanistic studies, development of new and extended technologies, the applications in animals, plants and the therapeutics.
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Nanoparticle-based CRISPR/Cas Delivery: An Emerging Tactic for Cancer Therapy.
Genome editing arose as a new promising approach for treating numerous intricate ailm ents including cancer. Over the past couple of decades, delivery technologies that have serendipitously been developed using viral vectors are successful to some extent in protein and nucleic acid delivery but their effectiveness still lags due to their efficiency, tissue targeting capabilities, and toxicity which must be further improved. With the infiltration of nanotechnology into every sphere of life, nano-vehicles can be implemented as an ideal modality that can overcome challenges, also can be introspective as new genome editing tools for cancer therapy owing to the safety and efficiency in clinical settings. Such projected substitution can help in developing highly efficacious therapy regimes which are successful in clinical settings. This emerging approach of incorporation of genome editors (CRISPR/Cas) in different nano vehicles and their utility in targeting various aspects of cancer therapy like treatment, diagnostics, modelling has been comprehensively done in this review.
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Survival and Evolution of CRISPR-Cas System in Prokaryotes and Its Applications.
Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution. Also, due to their diverse nature of functionality, cas9 endoribonuclease can be easily reprogrammed with the help of guide RNAs, showing unprecedented potential and significance for gene editing in treating genetic diseases. Here, we also discuss the use of NgAgo-gDNA system in genome editing of human cells.
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The case for banning heritable genome editing.
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Gene Editing: A Tool for Tackling Cephalopod Biology.
Cephalopods fascinate us but have been out of the reach of experimental manipulations at the genetic level. A new study describes editing of a gene in a squid using CRISPR.
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Cellular Inflammation-Induced Cleavage of Phosphorothioate DNA Locker Activates CRISPR/Cas9 Regulator for Gene Editing.
The CRISPR technology is a highly promising strategy for developing a versatile toolbox to engineer genetic circuits. However, achieving precise and specific control over the activity of the CRISPR/Cas9 system in response to intracellular processes remains a challenging endeavor. In this study, we present a cellular inflammation-induced activation of an engineered CRISPR/Cas9 regulator for gene regulation. A phosphorothioate (PS)-modified DNA sequence, referred as the "locker," is employed to deactivate single guide RNA (sgRNA), whose locker sequence complements the spacer region of sgRNA. In the presence of myeloperoxidase during cellular inflammation, a halogenation process is triggered, leading to the generation of HClO, specifically cleaving the PS site of locker and activating CRISPR/Cas9 for gene editing. The target GFP gene has been successfully edited, downregulating the GFP protein expression in HeLa cells. This study provides valuable insights into the CRISPR-based gene regulation through specific endogenous processes.
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Chemistry, manufacturing and controls for gene modified hematopoietic stem cells.
Gene modification of hematopoietic stem cells is increasingly becoming popular as a therapeutic approach, given the recent approvals and the number of new applications for clinical trials targeting monogenetic and immunodeficiency disorders. Technological advances in stem cell selection, culture, transduction and gene editing now allow for efficient ex vivo genetic manipulation of stem cells. Gene-addition techniques using viral vectors (mainly retrovirus- and lentivirus-based) and gene editing using various targeted nuclease platforms (e.g., Zinc finger, TALEN and Crispr/Cas9) are being applied to the treatment of multiple genetic and immunodeficiency disorders. Herein, the current state of the art in manufacturing and critical assays that are required for ex vivo manipulation of stem cells are addressed. Important quality control and safety assays that need to be planned early in the process development phase of these products for regulatory approval are also highlighted.
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Human genome editing: ask whether, not how.
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Novel application of ribonucleoprotein-mediated CRISPR-Cas9 gene editing in plant pathogenic oomycete species.
CRISPR-Cas9 gene editing has become an important tool for the study of plant pathogens, allowing researchers to functionally characterize specific genes involved in phytopathogenicity, virulence, and fungicide resistance. Protocols for CRISPR-Cas9 gene editing have already been developed for Phytophthoras, an important group of oomycete plant pathogens; however, these efforts have exclusively focused on agricultural pathosystems, with research lacking for forest pathosystems. We sought to develop CRISPR-Cas9 gene editing in two forest pathogenic Phytophthoras, Phytophthora cactorum and P. ramorum, using a plasmid-ribonucleoprotein (RNP) co-transformation approach. Our gene target in both species was the ortholog of PcORP1, which encodes an oxysterol-binding protein that is the target of the fungicide oxathiapiprolin in the agricultural pathogen P. capsici. We delivered liposome complexes, each containing plasmid DNA and CRISPR-Cas9 RNPs, to Phytophthora protoplasts using a polyethylene glycol-mediated transformation protocol. We obtained two ORP1 mutants in P. cactorum but were unable to obtain any mutants in P. ramorum. The two P. cactorum mutants exhibited decreased resistance to oxathiapiprolin, as measured by their radial growth relative to wild-type cultures on oxathiapiprolin-supplemented medium. Our results demonstrate the potential for RNP-mediated CRISPR-Cas9 gene editing in P. cactorum and provide a foundation for future optimization of our protocol in other forest pathogenic Phytophthora species.IMPORTANCECRISPR-Cas9 gene editing has become a valuable tool for characterizing the genetics driving virulence and pathogenicity in plant pathogens. CRISPR-Cas9 protocols are now well-established in several Phytophthora species, an oomycete genus with significant economic and ecological impact globally. These protocols, however, have been developed for agricultural Phytophthora pathogens only; CRISPR-Cas9 systems have not yet been developed for any forest pathogenic Phytophthoras. In this study, we sought to establish CRISPR-Cas9 gene editing in two forest Phytophthora pathogens that cause widespread tree mortality: P. cactorum and P. ramorum. We successfully obtained gene mutations in P. cactorum and demonstrated a decrease in fungicide resistance, a trait that could impact the pathogen's ability to cause disease. However, the same protocol did not yield any mutants in P. ramorum. The results of our study will serve as a baseline for the development of CRISPR-Cas9 gene editing in forest Phytophthoras and other oomycetes.
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A CRISPR-Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in Starmerella bombicola.
Gene editing technology plays an extremely significant role in synthetic biology and metabolic engineering. Traditional genetic manipulation methods, such as homologous recombination, however, are inefficient, time-consuming, and barely feasible when disrupting multiple genes simultaneously. Starmerella bombicola, a nonconventional yeast that overproduces sophorolipids, lacks convenient genetic tools for engineering strains. Here, we developed an efficient CRISPR-Cas9 genome editing technology by combining molecular element mining and expression system optimization for S. bombicola. This CRISPR-Cas9 system improved the efficiency of gene-integration/target gene-introducing disruption by homology-directed repair and realized the multi-gene simultaneous disruptions. Based on this CRISPR-Cas9 system, we also further constructed an engineered strain via the in vivo assembly of multiple DNA fragments (10 kb) that can produce acid-type sophorolipids. These results showed that the CRISPR-Cas9 system may be an efficient and convenient strategy to perform genetic manipulation in S. bombicola.
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