Браузер не даёт сохранять данные (например, приватный режим): закладки пропадут после закрытия вкладки.
Поиск статей PubMed
Ключевые слова, автор, журнал, годы — результаты из PubMed, аннотации с переводом на русский, закладки в браузере.
Найдено: 26 149
Страница 577 из 1000-
Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza.
CRISPR/Cas9 is a powerful genome editing tool that has been extensively used in model plants and crops, such as Arabidopsis thaliana, rice, wheat, and soybean. Here, we report the use of CRISPR/Cas9 to precisely knock out the committed diterpene synthase gene (SmCPS1) involved in tanshinone biosynthesis in Salvia miltiorrhiza, a traditional Chinese medicinal herb with significant pharmacological activities, such as vasorelaxation, protection against ischemia-reperfusion injury, and antiarrhythmic effects. Three homozygous and eight chimeric mutants were obtained from 26 independent transgenic hairy root lines by Agrobacterium rhizogenes-mediated transformation. The metabolomic analysis based on LC-qTOF-MS and Q-TRAP-LC-MS/MS revealed that tanshinones, especially cryptotanshinone, tanshinone IIA and tanshinone I, are completely missing in homozygous mutants, without influencing other phenolic acid metabolites. By contrast, tanshinones are decreased but still detectable in chimeric mutants, which is similar to a previously-reported an RNAi study of SmCPS1. These results demonstrate that Agrobacterium rhizogenes- mediated transformation using CRISPR/Cas9 is a simple and efficient genome editing tool in S. miltiorrhiza, thus paving the way for large-scale genome editing in S. miltiorrhiza, which is important for pathway elucidation of secondary metabolites, quality improvement, and yield increases for this valuable traditional Chinese medicinal herb.
-
Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells.
Prime editors consisting of Cas9-nickase and reverse transcriptase enable targeted precise editing of small DNA pieces, including all 12 kinds of base substitutions, insertions and deletions, while without requiring double-strand breaks or donor templates. Current optimized prime editing strategy (PE3) uses two guide RNAs to guide the performance of prime editor. One guide RNA carrying both spacer and templating sequences (pegRNA) guides prime editor to produce ssDNA break and subsequent extension, and the other one produces a nick in the complementary strand. Here, we demonstrated that positioning the nick sgRNA nearby the templating sequences of the pegRNA facilitated targeted large fragment deletion and that engineering both guide RNAs to be pegRNAs to achieve bi-direction prime editing (Bi-PE) further increase the efficiency by up to 16 times and improved the accuracy of editing products by 60 times. In addition, we showed that Bi-PE strategy also increased the efficiency of simultaneous conversion of multiple bases but not single base conversion over PE3. In conclusion, Bi-PE strategy expanded the editing scope and improved the efficiency and the accuracy of prime editing system, which might have a wide range of potential applications.
-
Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus.
CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5'-AAA-3' protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.
-
Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications.
The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins constitute the innate adaptive immune system in several bacteria and archaea. This immune system helps them in resisting the invasion of phages and foreign DNA by providing sequence-specific acquired immunity. Owing to the numerous advantages such as ease of use, low cost, high efficiency, good accuracy, and a diverse range of applications, the CRISPR-Cas system has become the most widely used genome editing technology. Hence, the advent of the CRISPR/Cas technology highlights a tremendous potential in clinical diagnosis and could become a powerful asset for modern medicine. This study reviews the recently reported application platforms for screening, diagnosis, and treatment of different diseases based on CRISPR/Cas systems. The limitations, current challenges, and future prospectus are summarized; this article would be a valuable reference for future genome-editing practices.
-
Search-and-remove genome editing allows selection of cells by DNA sequence.
The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However, such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here, we present SNIPE, a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells, which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations.
-
Systematic Comparison of Computational Tools for Sanger Sequencing-Based Genome Editing Analysis.
Successful genome editing depends on the cleavage efficiency of programmable nucleases (PNs) such as the CRISPR-Cas system. Various methods have been developed to assess the efficiency of PNs, most of which estimate the occurrence of indels caused by PN-induced double-strand breaks. In these methods, PN genomic target sites are amplified through PCR, and the resulting PCR products are subsequently analyzed using Sanger sequencing, high-throughput sequencing, or mismatch detection assays. Among these methods, Sanger sequencing of PCR products followed by indel analysis using online web tools has gained popularity due to its user-friendly nature. This approach estimates indel frequencies by computationally analyzing sequencing trace data. However, the accuracy of these computational tools remains uncertain. In this study, we compared the performance of four web tools, TIDE, ICE, DECODR, and SeqScreener, using artificial sequencing templates with predetermined indels. Our results demonstrated that these tools were able to estimate indel frequency with acceptable accuracy when the indels were simple and contained only a few base changes. However, the estimated values became more variable among the tools when the sequencing templates contained more complex indels or knock-in sequences. Moreover, although these tools effectively estimated the net indel sizes, their capability to deconvolute indel sequences exhibited variability with certain limitations. These findings underscore the importance of judiciously selecting and using an appropriate tool with caution, depending on the type of genome editing being performed.
-
An innovative approach using CRISPR-ribonucleoprotein packaged in virus-like particles to generate genetically engineered mouse models.
Genetically engineered mouse models (GEMMs) are crucial for investigating disease mechanisms, developing therapeutic strategies, and advancing fundamental biological research. While CRISPR gene editing has greatly facilitated the creation of these models, existing techniques still present technical challenges and efficiency limitations. Here, we establish a CRISPR-VLP-induced targeted mutagenesis (CRISPR-VIM) strategy, enabling precise genome editing by co-culturing zygotes with virus-like particle (VLP)-delivered gene editing ribonucleoproteins (RNPs) without requiring physical manipulation or causing cellular damage. We generate Plin1- and Tyr-knockout mice through VLP-based SpCas9 or adenine base editor (ABE)/sgRNA RNPs and characterize their phenotype and germline transmission. Additionally, we demonstrate cytosine base editor (CBE)/sgRNA-based C-to-T substitution or SpCas9/sgRNA-based knock-in using VLPs. This method further simplifies and accelerates GEMM generation without specialized techniques or equipment. Consequently, the CRISPR-VIM method can facilitate mouse modeling and be applied in various research fields.
-
Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins.
CRISPR loci and Cas proteins provide adaptive immunity in prokaryotes against invading bacteriophages and plasmids. In response, bacteriophages have evolved a broad spectrum of anti-CRISPR proteins (anti-CRISPRs) to counteract and overcome this immunity pathway. Numerous anti-CRISPRs have been identified to date, which suppress single-subunit Cas effectors (in CRISPR class 2, type II, V and VI systems) and multisubunit Cascade effectors (in CRISPR class 1, type I and III systems). Crystallography and cryo-electron microscopy structural studies of anti-CRISPRs bound to effector complexes, complemented by functional experiments in vitro and in vivo, have identified four major CRISPR-Cas suppression mechanisms: inhibition of CRISPR-Cas complex assembly, blocking of target binding, prevention of target cleavage, and degradation of cyclic oligonucleotide signalling molecules. In this Review, we discuss novel mechanistic insights into anti-CRISPR function that have emerged from X-ray crystallography and cryo-electron microscopy studies, and how these structures in combination with function studies provide valuable tools for the ever-growing CRISPR-Cas biotechnology toolbox, to be used for precise and robust genome editing and other applications.
-
Novel miR-29b target regulation patterns are revealed in two different cell lines.
MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct expression levels of miRNAs, including miR-29b, have been detected in various biological fluids and tissues from a large variety of disease models. However, how miRNAs "react" and function in different cellular environments is still largely unknown. In this study, the regulation patterns of miR-29b between human and mouse cell lines were compared for the first time. CRISPR/Cas9 gene editing was used to stably knockdown miR-29b in human cancer HeLa cells and mouse fibroblast NIH/3T3 cells with minimum off-targets. Genome editing revealed mir-29b-1, other than mir-29b-2, to be the main source of generating mature miR-29b. The editing of miR-29b decreased expression levels of its family members miR-29a/c via changing the tertiary structures of surrounding nucleotides. Comparing transcriptome profiles of human and mouse cell lines, miR-29b displayed common regulation pathways involving distinct downstream targets in macromolecular complex assembly, cell cycle regulation, and Wnt and PI3K-Akt signalling pathways; miR-29b also demonstrated specific functions reflecting cell characteristics, including fibrosis and neuronal regulations in NIH/3T3 cells and tumorigenesis and cellular senescence in HeLa cells.
-
The Complete Genome of Emcibacter congregatus ZYLT, a Marine Bacterium Encoding a CRISPR-Cas 9 Immune System.
Emcibacter congregatus ZYLT was isolated from a sediment sample cultured in situ in a coast located in the East China Sea. The genome of E. congregatus ZYLT was sequenced and assembled into one single circular chromosome with the size of 4,189,011 bp and G+C content of 52.6%. Genomic annotation showed that E. congregatus ZYLT had an intact Type II-C CRISPR-Cas system consists of three cas genes (cas 9, cas 1, and cas 2), 34 direct repeat sequences with the length of 36 bp, and 33 spacers. The predicted Cas 9 protein was smaller than most of existing genome editing tools. This structure might have potential in developing new gene editing system and uncovering the regulatory mechanisms of CRISPR-Cas system. Besides, the comparison between E. congregatus ZYLT and its relative species living in neritic environments unraveled some common traits of the defective strategies of these bacteria to face inshore challenges including the motility, multidrug resistance, and universal efflux pumps.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.