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Found: 26 179
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Enhancement of Free Fatty Acids Production in Rhodotorula toruloides Using the CRISPR/Cas9-Based Base Editor.
Rhodotorula toruloides is a promising cell factory to produce various value-added chemicals, including fatty acid derivatives. However, their metabolic engineering development has been hindered by the limited availability of genetic tools. In this study, an accurate and specific gene-editing tool, CRISPR/Cas-based cytidine base editor (CBE) system, was developed for the first time in R. toruloides to broaden its genetic toolbox. The target gene was disrupted by introducing a premature stop codon via C to T mutation. This system achieved single-gene disruption efficiencies of up to 90% and successfully disrupted four genes in parallel with 5% efficiency, marking a breakthrough in multiplexed editing for this yeast. To enable iterative engineering, an inducible Cre-loxP system was integrated, achieving an over 70% selection marker recycling efficiency. Application of this system enabled the construction of uracil-auxotrophic strains. Furthermore, the CBE system was employed to disrupt four genes involved in lipid metabolism, resulting in an engineered strain capable of producing 512.3 mg/L of free fatty acids, thereby demonstrating the utility of the CBE system as an efficient genome editing tool in R. toruloides. The study provides valuable tools to expand the genetic toolbox of R. toruloides and paves the way for fully exploiting its metabolic engineering potential.
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Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure.
Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE's base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE's indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites.
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The use of CRISPR/Cas9-based gene editing strategies to explore cancer gene function in mice.
CRISPR/Cas9 systems have revolutionised the field of gene editing, allowing for precise modifications to be generated in vivo to mimic the genetic events found in human cancer cells. These systems may be used to generate germline or somatic loss-of-function of events, and also chromosomal rearrangements, either constitutively or in a spatiotemporally controlled manner. Forward genetic screens have also been performed using CRISPR/Cas9 systems to identify new driver genes and approaches using catalytically inactive Cas9 fused to base editors have enabled genome editing with single-base precision. Here we discuss the many 'flavours' of the CRISPR/Cas9 system and give examples of their use for the generation of clinically-relevant mouse models of cancer.
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Prime editing-installed suppressor tRNAs for disease-agnostic genome editing.
Precise genome-editing technologies such as base editing1,2 and prime editing3 can correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. For diseases that are caused by premature stop codons, suppressor tRNAs (sup-tRNAs) offer a more general strategy. Existing approaches to use sup-tRNAs therapeutically, however, require lifelong administration4,5 or show modest potency, necessitating potentially toxic overexpression. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA. Iterative screening of thousands of variants of all 418 human tRNAs identified tRNAs with the strongest sup-tRNA potential. We optimized prime editing agents to install an engineered sup-tRNA at a single genomic locus without overexpression and observed efficient readthrough of premature termination codons and protein rescue in human cell models of Batten disease, Tay-Sachs disease and cystic fibrosis. In vivo delivery of a single prime editor that converts an endogenous mouse tRNA into a sup-tRNA extensively rescued disease pathology in a model of Hurler syndrome. PERT did not induce detected readthrough of natural stop codons or cause significant transcriptomic or proteomic changes. Our findings suggest the potential of disease-agnostic therapeutic genome-editing approaches that require only a single composition of matter to treat diverse genetic diseases.
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RNA recording in single bacterial cells using reprogrammed tracrRNAs.
Capturing an individual cell's transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected cellular transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed to base pair with sensed transcripts, converting them into guide RNAs. The guide RNAs then direct a Cas9 base editor to target an introduced DNA target. The extent of base editing can then be read in the future by sequencing. We use this approach, called TIGER (transcribed RNAs inferred by genetically encoded records), to record heterologous and endogenous transcripts in individual bacterial cells. TIGER can quantify relative expression, distinguish single-nucleotide differences, record multiple transcripts simultaneously and read out single-cell phenomena. We further apply TIGER to record metabolic bet hedging and antibiotic resistance mobilization in Escherichia coli as well as host cell invasion by Salmonella. Through RNA recording, TIGER connects current cellular states with past transcriptional states to decipher complex cellular responses in single cells.
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Updates on CRISPR-based gene editing in HIV-1/AIDS therapy.
Although tremendous efforts have been made to prevent and treat HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. The combination antiretroviral therapy (cART), although able to suppress HIV-1 replication, cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects. In recent years, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection. However, new gene-targeting tools derived from or functioning like CRISPR/Cas9, including base editor, prime editing, SHERLOCK, DETECTR, PAC-MAN, ABACAS, pfAGO, have been developed and optimized for pathogens detection and diseases correction. Here, we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection. We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future. Moreover, we discuss the caveats and problems that should be addressed before the clinical use of these versatile CRISPR-based gene targeting tools. Finally, we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.
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Artificial Intelligence in CRISPR-Cas Systems: A Review of Tool Applications.
Genetic engineering is a method used to alter an organism's DNA, which could entail altering a base pair, removing a section of DNA, or introducing a new DNA segment. Over time, genetic engineering has progressed from basic cloning for research purposes to advanced synthetic biology, leading to new biomedical applications. Targeted genomic editing is one method of cellular reprogramming that aims to change the state of a cell. The invention of CRISPR Cas systems has greatly simplified gene editing. These systems use a unique RNA-guided DNA endonuclease, a protein that can cut DNA and be trained to target new places by changing the sequence of its guide RNA. Integrating CRISPR-Cas systems with artificial intelligence opens new insights into the study of genetic engineering and its applications. Extensive research utilizing deep learning and machine learning has been conducted to predict the outcomes of CRISPR-Cas9 editing. Artificial intelligence also predicts RNA editing events and CRISPR off-target cleavage sites. Scientists often struggle to identify the ideal perturbation for their specific application because of the ample search space and expensive genetic trials. The algorithmic method using artificial intelligence utilizes the cause-and-effect link between variables in a complicated system like genome regulation to determine which perturbation is most effective in each successive round of testing, thereby making artificial intelligence an effective technique in gene editing.
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Applying CRISPR-Cas9 Genome Editing to Study Genes Involved in Peroxisome Biogenesis or Peroxisomal Functions.
The development and application of the CRISPR-Cas9 technology for genome editing of mammalian cells have opened up a wealth of possibilities for genetically modifying and manipulating human cells, and use in functional studies or therapeutic approaches.Here we describe the approach that we have been using successfully to generate multiple human cell lines with targeted (partial) gene deletions, i.e., knockout cells, or human cells with modified genomic nucleotide sequences, i.e., knock-in cells, in genes encoding known or putative proteins involved in peroxisome biogenesis or peroxisomal functions.
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A new and promising application of gene editing: CRISPR-controlled smart materials for tissue engineering, bioelectronics, and diagnostics.
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Transforming Plant Viruses into Vectors for Next-Generation Agriculture-A Review.
Plant viral vectors have evolved from tools for transient gene expression into a versatile platform for precise genetic intervention, offering a rapid, transgene-free alternative to conventional crop transformation. This review critically assesses their engineering for scalable field application, moving beyond foundational techniques like virus-induced gene silencing (VIGS). We highlight how advanced vector design, including deconstructed genomes and synthetic regulatory circuits enhances cargo capacity, specificity, and biosafety. The integration of viral delivery with CRISPR-Cas systems has unlocked virus-induced genome editing (VIGE), base editing, and prime editing, enabling heritable trait modification without tissue culture. However, the transition from proof-of-concept in model plants to robust field technology hinges on overcoming critical bottlenecks: expanding host range through chimeric vectors, ensuring environmental containment, and developing scalable delivery methods such as nano-formulations or adjusted agroinfiltration protocols. We evaluate these delivery routes and emerging synergies with nanobiotechnology for targeted and efficient applications. While challenges in regulation, public perception, and large-scale production persist, the strategic engineering of viral vectors for stability, specificity, and safety positions them as a transformative, next-generation biotechnological input for achieving sustainable crop improvement and protection under changing climatic conditions.
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