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: 26 157
Страница 606 из 1000-
Genome-wide CRISPR off-target prediction and optimization using RNA-DNA interaction fingerprints.
The powerful CRISPR genome editing system is hindered by its off-target effects, and existing computational tools achieved limited performance in genome-wide off-target prediction due to the lack of deep understanding of the CRISPR molecular mechanism. In this study, we propose to incorporate molecular dynamics (MD) simulations in the computational analysis of CRISPR system, and present CRISOT, an integrated tool suite containing four related modules, i.e., CRISOT-FP, CRISOT-Score, CRISOT-Spec, CRISORT-Opti for RNA-DNA molecular interaction fingerprint generation, genome-wide CRISPR off-target prediction, sgRNA specificity evaluation and sgRNA optimization of Cas9 system respectively. Our comprehensive computational and experimental tests reveal that CRISOT outperforms existing tools with extensive in silico validations and proof-of-concept experimental validations. In addition, CRISOT shows potential in accurately predicting off-target effects of the base editors and prime editors, indicating that the derived RNA-DNA molecular interaction fingerprint captures the underlying mechanisms of RNA-DNA interaction among distinct CRISPR systems. Collectively, CRISOT provides an efficient and generalizable framework for genome-wide CRISPR off-target prediction, evaluation and sgRNA optimization for improved targeting specificity in CRISPR genome editing.
-
Hydrazone chemistry-mediated CRISPR/Cas12a system for bacterial analysis.
In this study, a hydrazone chemistry-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system has been proposed for the fist time and constructed. In our system, hydrazone chemistry is designed and employed to accelerate the formation of a whole activation strand by taking advantage of the proximity effect induced by complementary base pairing, thus activating the CRISPR/Cas12a system quickly and efficiently. Moreover, the introduction of hydrazone chemistry can improve the specificity of the CRISPR/Cas12a system, allowing it to effectively distinguish single-base mismatches. The established system has been further applied to analyze Pseudomonas aeruginosa by specific recognition of the probe strand with a characteristic fragment in 16S rDNA to release the hydrazine group-modified activation strand. The method shows a wide linear range from 3.8 × 102 colony-forming units (CFU)/ml to 3.8 × 106 CFU/ml, with the lowest detection limit of 24 CFU/ml. Therefore, the introduction of hydrazone chemistry may also broaden the application of the CRISPR/Cas12a system.
-
Analysis of CRISPR-Cas System in Streptococcus thermophilus and Its Application.
CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) loci, which provide a specific immunity against exogenous elements, are hypervariable among distinct prokaryotes. Based on previous researches, this review focuses on concluding systematical genome editing protocols in Streptococcus thermophilus. Firstly, its protocols and optimized conditions in gene editing are introduced. What's more, classification and diversity analyses of S. thermophilus CRISPR-Cas benefit the further understanding of evolution relationship among Streptococcus. Ability of its foreign segment integration and spacer source analyses also indicate a new direction of phage resistance. Above all, all of these point out its potential to be regarded as another model system other than type II CRISPR-Cas in Streptococcus pyogenes.
-
TALEN-Based Chemically Inducible, Dimerization-Dependent, Sequence-Specific Nucleases.
For precise genome editing, it is important to control the activity of sequence-specific nucleases. We have constructed a chemically inducible nuclease system based on the dimerization of FKBP and FRB domains in the presence of rapamycin and designated it as a chemically inducible dimerization (CID). The CID was designed to occur at the interlinker section between DNA binding domains and the FokI catalytic domain. Thus, induction of cleavage should occur quickly after addition of rapamycin because components of proteins are already in active form and located in the nucleus. This CID-dependent sequence-specific nuclease has potential to be applied for time-resolved analysis of the mutation induction mechanism in the genome.
-
Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models.
Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.
-
Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility.
CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N4AH and N5HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N3VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.
-
A Serum Resistant Polymer with Exceptional Endosomal Escape and mRNA Delivery Efficacy for CRISPR Gene Therapy.
Nanoparticle-based mRNA delivery offers a versatile platform for innovative therapies. However, most of the current delivery systems are limited by poor serum tolerance, suboptimal endosomal escape and mRNA delivery efficacy. Herein, a highly efficient mRNA-delivering material is identified from a library of fluoropolymers. The lead material FD17 shows exceptional serum stability and endosomal escape, enabling efficient mRNA delivery into various cell types, surpassing commercial mRNA delivery reagents such as Lipofectamine 3000. The formed mRNA nanoparticles adsorb abundant serum albumin on the surface, which facilitates cellular uptake via scavenger receptor-mediated endocytosis. FD17 enables the delivery of mRNAs encoding CRE, Cas9, and base editor hyCBE for efficient genome editing. The material mediates CRISPR/Cas9 gene therapy via intraocular injection effectively down-regulates vascular endothelial growth factor A in retinal pigment epithelial cells of mice, yielding promising therapeutic responses against laser-induced choroidal neovascularization. The discovered material in this study shows great promise for the development of mRNA therapeutics to combat a wide range of diseases.
-
Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome.
RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, the nontrivial level of off-target activity reported in several human cells may hamper clinical translation. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We hypothesized that more stringent PAM requirements reduce the occurrence of off-target mutagenesis. To test this postulation, we generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, PRKDC and CARD11. Our results demonstrate that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved. In conclusion, our results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications.
-
Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision.
We developed a CRISPR-Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. We validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply our method to improve tolerance to growth inhibitors.
-
CRISPR/Cas9 editing of the MYO7A gene in rhesus macaque embryos to generate a primate model of Usher syndrome type 1B.
Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild-type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.