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Found: 26 179
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Circular RNA-mediated inverse prime editing in human cells.
Prime editors are restricted to performing precise edits downstream of cleavage sites, thereby limiting their editing scope. Therefore, we develop inverse prime editors (iPEs) that act upstream of the nickase cleavage site by replacing nCas9-H840A with nCas9-D10A, but the editing efficiencies are limited. To address this limitation, we develop circular RNA-mediated iPEs (ciPEs), achieving editing efficiencies ranging from 0.1% to 24.7%. Further optimization using Rep-X helicase increases editing efficiencies to a range of 2.7%-55.4%. The Rep-X-assisted ciPE system thus expands the scope of editing and improves efficiencies at genomic sites that are previously difficult to target. The Rep-X-assisted ciPE system will complement canonical PE system in enabling more extensive and efficient editing across a wider range of the human genome.
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Gene drives gaining speed.
Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR-Cas9-based gene-drive systems are highly efficient in laboratory settings, offering the potential to reduce the prevalence of vector-borne diseases, crop pests and non-native invasive species. However, concerns have been raised regarding the potential unintended impacts of gene-drive systems. This Review summarizes the phenomenal progress in this field, focusing on optimal design features for full-drive elements (drives with linked Cas9 and guide RNA components) that either suppress target mosquito populations or modify them to prevent pathogen transmission, allelic drives for updating genetic elements, mitigating strategies including trans-complementing split-drives and genetic neutralizing elements, and the adaptation of drive technology to other organisms. These scientific advances, combined with ethical and social considerations, will facilitate the transparent and responsible advancement of these technologies towards field implementation.
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Methods for characterizing, applying, and teaching CRISPR-Cas systems.
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Computer-aided engineering of CRISPR-Cas proteins for enhanced human genome editing.
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A CRISPR-Cas Cure for HIV/AIDS.
Human immunodeficiency virus (HIV) infections and HIV-induced acquired immunodeficiency syndrome (AIDS) continue to represent a global health burden. There is currently no effective vaccine, nor any cure, for HIV infections; existing antiretroviral therapy can suppress viral replication, but only as long as antiviral drugs are taken. HIV infects cells of the host immune system, and it can establish a long-lived viral reservoir, which can be targeted and edited through gene therapy. Gene editing platforms based on the clustered regularly interspaced palindromic repeat-Cas system (CRISPR-Cas) have been recognized as promising tools in the development of gene therapies for HIV infections. In this review, we evaluate the current landscape of CRISPR-Cas-based therapies against HIV, with an emphasis on the infection biology of the virus as well as the activity of host restriction factors. We discuss the potential of a combined CRISPR-Cas approach that targets host and viral genes to activate antiviral host factors and inhibit viral replication simultaneously. Lastly, we focus on the challenges and potential solutions of CRISPR-Cas gene editing approaches in achieving an HIV cure.
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Enhancing the quality of staple food crops through CRISPR/Cas-mediated site-directed mutagenesis.
The enhancement of CRISPR-Cas gene editing with robust nuclease activity promotes genetic modification of desirable agronomic traits, such as resistance to pathogens, drought tolerance, nutritional value, and yield-related traits in crops. The genetic diversity of food crops has reduced tremendously over the past twelve millennia due to plant domestication. This reduction presents significant challenges for the future especially considering the risks posed by global climate change to food production. While crops with improved phenotypes have been generated through crossbreeding, mutation breeding, and transgenic breeding over the years, improving phenotypic traits through precise genetic diversification has been challenging. The challenges are broadly associated with the randomness of genetic recombination and conventional mutagenesis. This review highlights how emerging gene-editing technologies reduce the burden and time necessary for developing desired traits in plants. Our focus is to provide readers with an overview of the advances in CRISPR-Cas-based genome editing for crop improvement. The use of CRISPR-Cas systems in generating genetic diversity to enhance the quality and nutritional value of staple food crops is discussed. We also outlined recent applications of CRISPR-Cas in developing pest-resistant crops and removing unwanted traits, such as allergenicity from crops. Genome editing tools continue to evolve and present unprecedented opportunities to enhance crop germplasm via precise mutations at the desired loci of the plant genome.
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Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step towards antivirulence therapy.
Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), driven by virulence factors such as iron acquisition systems and adhesive pili. In this study, we employed CRISPR-Cas9-mediated genome editing to functionally inactivate two critical virulence genes-iucD, involved in aerobactin-mediated iron uptake, and papC, encoding the outer membrane usher protein essential for P pilus assembly. Using a clinical UPEC isolate, we introduced premature stop codons via homologous repair templates guided by gene-specific single-guide RNAs. Colony PCR and Sanger sequencing confirmed precise site-specific editing, leading to truncated protein variants. In silico analyses using InterPro and Swiss-Model revealed a complete loss of essential domains in both proteins. Molecular docking studies demonstrated a marked reduction in binding affinities of truncated iucD for NAD(P)H and impaired protein-protein interaction between truncated PapC and PapG. This study highlights the utility of CRISPR-Cas9 as a powerful tool for dissecting bacterial pathogenesis and supports the potential of targeting virulence determinants like iucD and papC as part of an antivirulence strategy for managing UPEC infections.
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Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Cas12f1 nuclease.
Gene therapy is limited by inefficient delivery of large clustered regularly interspaced short palindromic repeat (CRISPR) effectors, such as Cas9 and Cas12a nucleases. Cas12f nucleases are currently one of the most compact CRISPR genome editors. However, the available toolkit of efficient Cas12f editors is limited. Here, we report the characterization and engineering of a miniature CRISPR-Cas12f system from Syntrophomonas palmitatica (SpaCas12f1, 497 amino acids). We show that CRISPR-SpaCas12f1 cleaves double-stranded DNA (dsDNA) with 5' T-rich PAM specificity and is naturally active for genome editing in bacteria. We identify that CRISPR-SpaCas12f1 trans-activating CRISPR RNA (tracrRNA) harbors a unique head-to-toe hairpin structure, and the natural hairpin structure is a key factor in restricting genome editing by SpaCas12f1 in human cells. Systematical engineering of SpaCas12f1 guide RNA transforms CRISPR-SpaCas12f1 into an efficient genome editor comparable to Francisella novicida CRISPR-Cas12a. Our findings expand the mini CRISPR toolbox, paving the way for therapeutic applications of CRISPR-SpaCas12f1 and engineering compact genome manipulation technologies.
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Microinjection for precision genome editing in Caenorhabditis elegans.
In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C. elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C. elegans, to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).
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Unlocking the Synthetic Potential of Yarrowia lipolytica: Innovating Gene Expression Tools.
Yarrowia lipolytica, with its robust lipid metabolism capabilities, efficient secretion system and generally recognised as safe (GRAS) status, has become a highly promising microbial chassis in synthetic biology. However, compared with model microorganisms such as Saccharomyces cerevisiae, the underdevelopment of gene expression tools in Y. lipolytica has become a critical bottleneck, limiting its industrial application. Currently, its core tools face two critical challenges: promoters with limited dynamic regulatory capacity, leading to metabolic flux imbalance; and gene editing systems plagued by low efficiency and poor multiplex compatibility. This opinion article focuses on these two pivotal directions to dissect their technical bottlenecks and propose innovative solutions: constructing dynamic transcriptional regulatory modules through machine learning guided design and synthetic biology approaches and developing orthogonal CRISPR systems and multiplex editing platforms.
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