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: 26 143
Страница 476 из 1000-
Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor.
-
One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces.
The RNA-guided DNA editing technology CRISPRs (clustered regularly interspaced short palindromic repeats)/Cas9 had been used to introduce double-stranded breaks into genomes and to direct subsequent site-specific insertions/deletions or the replacement of genetic material in bacteria, such as Escherichia coli, Streptococcus pneumonia, and Lactobacillus reuteri. In this study, we established a high-efficiency CRISPR/Cas9 genome editing plasmid pKCcas9dO for use in Streptomyces genetic manipulation, which comprises a target-specific guide RNA, a codon-optimized cas9, and two homology-directed repair templates. By delivering pKCcas9dO series editing plasmids into the model strain Streptomyces coelicolor M145, through one-step intergeneric transfer, we achieved the genome editing at different levels with high efficiencies of 60%-100%, including single gene deletion, such as actII-orf4, redD, and glnR, and single large-size gene cluster deletion, such as the antibiotic biosynthetic clusters of actinorhodin (ACT) (21.3 kb), undecylprodigiosin (RED) (31.6 kb), and Ca(2+)-dependent antibiotic (82.8 kb). Furthermore, we also realized simultaneous deletions of actII-orf4 and redD, and of the ACT and RED biosynthetic gene clusters with high efficiencies of 54% and 45%, respectively. Finally, we applied this system to introduce nucleotide point mutations into the rpsL gene, which conferred the mutants with resistance to streptomycin. Notably, using this system, the time required for one round of genome modification is reduced by one-third or one-half of those for conventional methods. These results clearly indicate that the established CRISPR/Cas9 genome editing system substantially improves the genome editing efficiency compared with the currently existing methods in Streptomyces, and it has promise for application to genome modification in other Actinomyces species.
-
Programmable RNA base editing with photoactivatable CRISPR-Cas13.
CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified and found to exhibit a low background and high inducibility. We observed significant light-induced perturbation of endogenous transcripts by paCas13. We further present a light-inducible base-editing system, herein called the padCas13 editor, by fusing ADAR2 to catalytically inactive paCas13 fragments. The padCas13 editor enabled reversible RNA editing under light and was effective in editing A-to-I and C-to-U RNA bases, targeting disease-relevant transcripts, and fine-tuning endogenous transcripts in mammalian cells in vitro. The padCas13 editor was also used to adjust post-translational modifications and demonstrated the ability to activate target transcripts in a mouse model in vivo. We therefore present a light-inducible RNA-modulating technique based on CRISPR-Cas13 that enables target RNAs to be diversely manipulated in vitro and in vivo, including through RNA degradation and base editing. The approach using the paCas13 system can be broadly applicable to manipulating RNA in various disease states and physiological processes, offering potential additional avenues for research and therapeutic development.
-
Development and Characterization of RNA Aptamer-Mediated Modular Base Editors Containing Staphylococcus aureus Cas9 Derivatives and Novel Deaminase Orthologs.
Base editing enables precise genome modifications without introducing DNA double-strand breaks. Using Streptococcus pyogenes Cas9 as a prototype, we previously developed a modular base editing platform in which the deaminase is recruited by an RNA aptamer engineered into the gRNA, thereby separating sequence recognition from base modification. Here, we expanded this modular base editor toolbox by engineering Staphylococcus aureus Cas9 (SaCas9) in combination with various vertebrate effectors derived from activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic subunit 1 (APOBEC1) orthologs, from bat, lizard, human, and rat. Moreover, we adopted the SaCas9 variants with different protospacer adjacent motif requirements. These base editors generally showed high editing efficiency with low on-target indel formation and low-to-undetectable off-target activities. Quantitative and qualitative differences in editing occur among the base editors when applied to diverse loci, allowing sequence-specific optimization. Together, our study demonstrates the effectiveness of the SaCas9 modular base editors, the robustness of the platform's modularity, and its feasibility for convenient screening of target-specific base editors.
-
Recent development in CRISPR-Cas systems for cardiac disease.
The CRISPR-Cas system has emerged as a revolutionary tool in genetic research, enabling highly precise gene editing and significantly advancing the field of cardiovascular science. This chapter provides a comprehensive overview of the latest developments in utilizing CRISPR-Cas technologies to investigate and treat heart diseases. It delves into the application of CRISPR-Cas9 for creating accurate models of complex cardiac conditions, such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and various arrhythmias, which are essential for understanding disease mechanisms and testing potential therapies. The therapeutic potential of gene editing is also explored, with a focus on genes like PCSK9 and ANGPTL3 that play critical roles in lipid metabolism and cardiovascular health, offering promising avenues for new treatments. Furthermore, the expanding applications of CRISPR in heart tissue regeneration are examined, which could revolutionize the repair of damaged heart tissue. Cutting-edge techniques such as base editing and prime editing are discussed, highlighting their potential to further refine genetic interventions. The discussion concludes by addressing the challenges associated with delivering CRISPR components efficiently and safely, while also exploring recent innovations that may overcome these hurdles, providing insights into the future directions of CRISPR technology in cardiovascular medicine.
-
CRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes.
Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols.
-
CRISPR/Cas-Mediated Resistance against Viruses in Plants.
CRISPR/Cas9 provides a robust and widely adaptable system with enormous potential for genome editing directed towards generating useful products. It has been used extensively to generate resistance against viruses infecting plants with more effective and prolonged efficiency as compared with previous antiviral approaches, thus holding promise to alleviate crop losses. In this review, we have discussed the reports of CRISPR/Cas-based virus resistance strategies against plant viruses. These strategies include approaches targeting single or multiple genes (or non-coding region) in the viral genome and targeting host factors essential for virus propagation. In addition, the utilization of base editing has been discussed to generate transgene-free plants resistant to viruses. This review also compares the efficiencies of these approaches. Finally, we discuss combinatorial approaches, including multiplexing, to increase editing efficiency and bypass the generation of escape mutants.
-
CRISPR DNA Base Editing Strategies for Treating Retinitis Pigmentosa Caused by Mutations in Rhodopsin.
Retinitis pigmentosa (RP) is the most common group of inherited retinal degenerations and pathogenic variants in the Rhodopsin (RHO) gene are major cause for autosomal dominant RP (adRP). Despite extensive attempts to treat RHO-associated adRP, standardized curative treatment is still lacking. Recently developed base editors offer an exciting opportunity to correct pathogenic single nucleotide variants and are currently able to correct all transition variants and some transversion variants. In this study, we analyzed previously reported pathogenic RHO variants (n = 247) for suitable PAM sites for currently available base editors utilizing the Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9) or the KKH variant of SaCas9 (KKH-SaCas9) to assess DNA base editing as a treatment option for RHO-associated adRP. As a result, 55% of all the analyzed variants could, in theory, be corrected with base editors, however, PAM sites were available for only 32% of them and unwanted bystander edits were predicted for the majority of the designed guide RNAs. As a conclusion, base editing offers exciting possibilities to treat RHO-associated adRP in the future, but further research is needed to develop base editing constructs that will provide available PAM sites for more variants and that will not introduce potentially harmful bystander edits.
-
A genome-wide base-editing screen uncovers a pivotal role of paxillin δ ubiquitination in influenza virus infection.
Dissecting host factors critical for viral infection and understanding their mechanisms of action is critical for identifying drug targets. Here, we leverage a genome-wide CRISPR base-editing screen to identify functional lysine residues in host factors required for influenza A virus (IAV) replication. Multiple host genes, including GSTM4, FLNC, HMGB1, ZNF236, GRIP1, and PXN, along with regulatory lysine codons, are identified. Among these, paxillin (encoded by PXN) is identified as an important host entry factor. Depletion of paxillin significantly reduces IAV infection in both cell cultures and mice. Further analysis suggests that the δ isoform of paxillin, rather than the canonical β isoform, plays the key role. Additionally, our data indicate that lysine 68 of paxillin δ undergoes K6-linked ubiquitination and regulates influenza virus replication via modulating endosome-dependent viral entry. These observations contribute to understanding how influenza viruses interact with host factors and may inform therapeutic development.
-
In vivo gene editing via homology-independent targeted integration for adrenoleukodystrophy treatment.
Adrenoleukodystrophy (ALD) is caused by various pathogenic mutations in the X-linked ABCD1 gene, which lead to metabolically abnormal accumulations of very long-chain fatty acids in many organs. However, curative treatment of ALD has not yet been achieved. To treat ALD, we applied two different gene-editing strategies, base editing and homology-independent targeted integration (HITI), in ALD patient-derived fibroblasts. Next, we performed in vivo HITI-mediated gene editing using AAV9 vectors delivered via intravenous administration in the ALD model mice. We found that the ABCD1 mRNA level was significantly increased in HITI-treated mice, and the plasma levels of C24:0-LysoPC (lysophosphatidylcholine) and C26:0-LysoPC, sensitive diagnostic markers for ALD, were significantly reduced. These results suggest that HITI-mediated mutant gene rescue could be a promising therapeutic strategy for human ALD treatment.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.