Your browser does not allow saving data (e.g. private mode): bookmarks will be lost when the tab closes.
Search PubMed
Keywords, author, journal, years — results from PubMed, abstracts with Russian translation, bookmarks in your browser.
Found: 26 107
Page 161 of 1000-
SWITCHER, a CRISPR-inducible floxed wild-type Cre regulating CRISPR activity.
Although several Cre-regulated CRISPR/Cas platforms exist, a CRISPR/Cas-controlled Cre-system remains a challenge. Here, we present a genetic switch we term SWITCHER based on a floxed wild-type Cre-construct representing a CRISPR-inducible and self-limiting kill switch. By leveraging CRISPR/Cas12a-mediated crRNA-array maturation, we showcase SWITCHER's dual role-not just as a recombinase but as a CRISPR switch, capable of orchestrating distinct Cas12a/crRNA-encoded programs.
-
Genetic toolkits for engineering Rhodococcus species with versatile applications.
Rhodococcus spp. are a group of non-model gram-positive bacteria with diverse catabolic activities and strong adaptive capabilities, which enable their wide application in whole-cell biocatalysis, environmental bioremediation, and lignocellulosic biomass conversion. Compared with model microorganisms, the engineering of Rhodococcus is challenging because of the lack of universal molecular tools, high genome GC content (61% ~ 71%), and low transformation and recombination efficiencies. Nevertheless, because of the high interest in Rhodococcus species for bioproduction, various genetic elements and engineering tools have been recently developed for Rhodococcus spp., including R. opacus, R. jostii, R. ruber, and R. erythropolis, leading to the expansion of the genetic toolkits for Rhodococcus engineering. In this article, we provide a comprehensive review of the important developed genetic elements for Rhodococcus, including shuttle vectors, promoters, antibiotic markers, ribosome binding sites, and reporter genes. In addition, we also summarize gene transfer techniques and strategies to improve transformation efficiency, as well as random and precise genome editing tools available for Rhodococcus, including transposition, homologous recombination, recombineering, and CRISPR/Cas9. We conclude by discussing future trends in Rhodococcus engineering. We expect that more synthetic and systems biology tools (such as multiplex genome editing, dynamic regulation, and genome-scale metabolic models) will be adapted and optimized for Rhodococcus.
-
CRISPR revolution: Unleashing precision pathogen detection to safeguard public health and food safety.
Foodborne pathogens represent a significant challenge to global food safety, causing widespread illnesses and economic losses. The growing complexity of food supply chains and the emergence of antimicrobial resistance necessitate rapid, sensitive, and portable diagnostic tools. CRISPR technology has emerged as a transformative solution, offering unparalleled precision and adaptability in pathogen detection. This review explores CRISPR's role in addressing critical gaps in traditional and modern diagnostic methods, emphasizing its advantages in sensitivity, specificity, and scalability. CRISPR-based diagnostics, such as Cas12 and Cas13 systems, enable rapid detection of bacterial and viral pathogens, as well as toxins and chemical hazards, directly in food matrices. Their integration with isothermal amplification techniques and portable biosensors enhances field applicability, making them ideal for decentralized and real-time testing. Additionally, CRISPR's potential extends beyond food safety, contributing to public health efforts by monitoring antimicrobial resistance and supporting One Health frameworks. Despite these advancements, challenges remain, including issues with performance in complex food matrices, scalability, and regulatory barriers. This review highlights future directions, including AI integration for assay optimization, the development of universal CRISPR platforms, and the adoption of sustainable diagnostic solutions. By tackling these challenges, CRISPR has the potential to redefine global food safety standards and create a more resilient food system. Collaborative research and innovation will be critical to fully unlocking its transformative potential in food safety and public health.
-
CRISPR System: A High-throughput Toolbox for Research and Treatment of Parkinson's Disease.
In recent years, the innovation of gene-editing tools such as the CRISPR/Cas9 system improves the translational gap of treatments mediated by gene therapy. The privileges of CRISPR/Cas9 such as working in living cells and organs candidate this technology for using in research and treatment of the central nervous system (CNS) disorders. Parkinson's disease (PD) is a common, debilitating, neurodegenerative disorder which occurs due to loss of dopaminergic neurons and is associated with progressive motor dysfunction. Knowledge about the pathophysiological basis of PD has altered the classification system of PD, which manifests in familial and sporadic forms. The first genetic linkage studies in PD demonstrated the involvement of Synuclein alpha (SNCA) mutations and SNCA genomic duplications in the pathogenesis of PD familial forms. Subsequent studies have also insinuated mutations in leucine repeat kinase-2 (LRRK2), Parkin, PTEN-induced putative kinase 1 (PINK1), as well as DJ-1 causing familial forms of PD. This review will attempt to discuss the structure, function, and development in genome editing mediated by CRISP/Cas9 system. Further, it describes the genes involved in the pathogenesis of PD and the pertinent alterations to them. We will pursue this line by delineating the PD linkage studies in which CRISPR system was employed. Finally, we will discuss the pros and cons of CRISPR employment vis-à-vis the process of genome editing in PD patients' iPSCs.
-
Markerless genome editing in Clostridium beijerinckii using the CRISPR-Cpf1 system.
CRISPR-Cpf1 is a type V CRISPR system that has recently been exploited for genome engineering purposes. Compared to the well-known Streptococcus pyogenes CRISPR-Cas9 system, the effector protein Cpf1 recognizes T-rich protospacer-adjacent motif (PAM) instead of G-rich PAM (used by CRISPR-Cas9), which could offer a substantial expansion of the existing genetic toolbox for genome editing. In this study, we report the implementation of the Acidaminococcus sp. Cpf1 (AsCpf1) for markerless genome engineering in Clostridium beijerinckii, a prominent species for biosolvent production through the well-known Acetone-Butanol-Ethanol (ABE) pathway. A lactose inducible promoter was used to control the expression of AsCpf1 to decrease its toxicity, while a constitutive small RNA promoter was employed to drive the expression of pre-crRNA. A One-Step-Assembly (OSA) approach was employed to construct the CRISPR-Cpf1-based vector in one single step, which simplified and streamlined the plasmid construction process. Using the customized CRISPR-Cpf1 system, we successfully deleted spo0A and pta genes in C. beijerinckii, with an editing efficiency of up to 100%. Altogether, our results demonstrated the easy programmability and high efficiency of the CRISPR-Cpf1 system for versatile genome engineering purposes. This study provides valuable guidance and essential references for repurposing the CRISPR-Cpf1 system for genome engineering in other microorganisms.
-
Yeast chromosomal engineering to improve industrially-relevant phenotypes.
Genome structural variations enable microbes to evolve quickly under environmental stress. Recent efforts in synthetic biology have shown the ability of yeast chromosomal engineering to generate a larger scale of genome structural variations, which require a high efficiency of DNA rearrangement technology. In this review, we summarize the recent development of the SCRaMbLE system, an evolutionary approach, and the CRISPR/Cas9 technology to generate yeast genome rearrangement. Both technologies exhibit the powerful applications of chromosomal engineering to accelerate phenotypic evolution. We highlight several studies where yeast genome rearrangement has successfully improved industrially-relevant phenotypes, including the production of novel medicine, nutrition supplements, anti-tumor molecules, and the tolerance of environmental stress and drug resistance.
-
"Perfect" designer chromosome V and behavior of a ring derivative.
Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in Saccharomyces cerevisiae under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.
-
A Potent CRISPR-Cas12l Double-Strand Break Gene Editor.
Recently, a new family of CRISPR-Cas12 endonucleases from an unexplored phylum of bacteria, Armatimonadota, was discovered. Named Cas12l, they are compact (800-900 aa), recognize a 5' C-rich protospacer adjacent motif, and present an N-terminal domain that stretches from the beginning to the end of the ribonucleoprotein-bound DNA target site, effectively locking it in place. Here, structure-guided rational design supplemented with AI-based large protein language model predictions was used to improve rates of DNA target cleavage of a family member, Asp2Cas12l. Compared to the wild-type, engineered variants exhibited an approximately 10-fold increase in double-strand break (DSB) editing efficiency in human cells with less target-to-target variation. Moreover, frequencies of editing were comparable to those of SpCas9 at overlapping target sites, and their DSBs efficiently corrected by homology-directed repair (39-56% of editing outcomes). Altogether, this study extends our understanding of CRISPR-Cas12 protein engineering and offers a potent new alternative for DSB-mediated genome editing in human cells.
-
CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery.
Epigenetics refers to chemical modifications of histone proteins and DNA that can regulate the expression of genes. The human epigenome is altered dynamically during cell differentiation and aging, and many diseases are associated with aberrant epigenome patterning. Recent advances in CRISPR have led to the development of programmable tools to edit epigenetic modifications at targeted genomic loci, enabling precise rewriting of epigenetic modifications in human cells. CRISPR-based epigenome editors rely on catalytically dead Cas9 coupled with epigenetic modifiers that ultimately result in programmed repression or activation of targeted genes in mammalian genomes. Unlike traditional genome editing methods, epigenome editing does not require DNA breaks or changes in the human genome sequence and thus serves as a safer alternative to control gene expression. In this protocol, we highlight two different methods to perform dCas9-mediated epigenome editing in human cell lines using plasmid DNA transfections and nucleofection of mRNAs encoding CRISPR epigenome editors. We demonstrate programmable epigenome editing to transiently repress genes using CRISPR interference (CRISPRi) and for silencing genes durably for many weeks using CRISPRoff, a fusion of dCas9 to the KRAB domain and de novo DNA methyltransferase complex. We also provide guidance on quantitative methods to measure successful epigenome editing of target genes and key considerations on which epigenome editing tool to use, depending on experimental criteria.
-
RNP-Based Control Systems for Genetic Circuits in Synthetic Biology Beyond CRISPR.
Ribonucleoproteins (RNPs) are RNA-protein complexes utilized natively in both prokaryotes and eukaryotes to regulate essential processes within the cell. Over the past few years, many of these native systems have been adapted to provide control over custom genetic targets. Engineered RNP-based control systems allow for fine-tune regulation of desired targets, by providing customizable nucleotide-nucleotide interactions. However, as there have been several engineered RNP systems developed recently, identifying an optimal system for various bioprocesses is challenging. Here, we review the most successful engineered RNP systems and their applications to survey the current state of the field. Additionally, we provide selection criteria to provide users a streamlined method for identifying an RNP control system most useful to their own work. Lastly, we discuss future applications of RNP control systems and how they can be utilized to address the current grand challenges of the synthetic biology community.
PubMed returns only the first 10,000 results — refine the query to see the rest.