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: 26 220
Страница 744 из 1000-
For China, a CRISPR first goes too far.
-
Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy.
Precise editing of the genome of a living body is a goal pursued by scientists in many fields. In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome-editing systems have become a revolutionary toolbox for gene editing across various species. However, the low transfection efficiency of the CRISPR/Cas9 system to mammalian cells in vitro and in vivo is a big obstacle hindering wide and deep application. In this review, recently developed delivery strategies for various CRISPR/Cas9 formulations and their applications in treating gene-related diseases are briefly summarized. This review should inspire others to explore more efficient strategies for CRISPR system delivery and gene therapy.
-
Seeking more nucleases.
-
A Variant of LbCas12a and Elevated Incubation Temperatures Enhance the Rate of Gene Editing in the Oomycete Phytophthora infestans.
CRISPR-Cas editing systems have proved to be powerful tools for functional genomics research, but their effectiveness in many non-model species remains limited. In the potato and tomato pathogen Phytophthora infestans, an editing system was previously developed that expresses the Lachnospiracae bacterium Cas12a endonuclease (LbCas12a) and guide RNA from a DNA vector. However, the method works at low efficiency. Based on a hypothesis that editing is constrained by a mismatch between the optimal temperatures for P. infestans growth and endonuclease catalysis, we tested two strategies that increased the frequency of editing of two target genes by about 10-fold. First, we found that editing was boosted by a mutation in LbCas12a (D156R) that had been reported to expand its catalytic activity over a broader temperature range. Second, we observed that editing was enhanced by transiently incubating transformed tissue at a higher temperature. These modifications should make CRISPR-Cas12a more useful for interrogating gene and protein function in P. infestans and its relatives, especially species that grow optimally at lower temperatures. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
-
CRISPR-Activation: Boosting Expression of Plant MIRs.
In the field of plant genome editing, the CRISPR/Cas system stands out as an exceptionally versatile tool. Originally conceived for gene editing, the catalytically inactive Cas9 variant, known as dead Cas9 (dCas9), takes on a new role when coupled with transcriptional effectors, offering a finely tuned mechanism for controlling gene expression. This innovation has paved the way for the evolution of sophisticated systems such as CRISPR-Act 2.0 and CRISPR-Act 3.0, which significantly enhance transcriptional activation in plants. This chapter serves as an introduction to a protocol that harnesses CRISPR-activation (CRISPRa) to modulate the transcription of plant miRNA genes. Recognizing the imperative for meticulous construct design and promoter identification, the protocol outlines steps encompassing vector construction. It underscores the critical importance of comprehending miRNA promoter regions and cis-acting elements, presenting a holistic approach for effectively employing CRISPRa in manipulating miRNA expression to modulate traits in plants.
-
CRISPR-Cpf1-Mediated T4 Phage Genome Editing for One-Step In Vivo Display of Heterologous Protein.
The T4 phage is a robust vector for high-density heterologous protein display. It leverages two non-essential outer capsid proteins, i.e., Soc (~870 copies) and Hoc (~155 copies). These two proteins enable the efficient display of target proteins on the capsid of T4. Here, we detail the workflow for one-step in vivo display of a heterologous protein. Specifically, this method utilizes CRISPR-Cpf1-mediated gene editing technology to insert the sequence of interest (using mCherry as an example) downstream of the Soc encoding gene within the T4 phage genome, resulting in a Soc-fused recombinant protein. This engineering method allows for endogenous expression of the Soc-mCherry recombinant protein within Escherichia coli cells during phage replication, after which the recombinant protein spontaneously assembles onto the capsid of the engineered phage. By providing a universal framework suitable for in vivo display, this approach empowers researchers to readily construct tailored T4 nanoparticles for diverse biotechnological applications.
-
Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing.
Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called 'BALL', enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.
-
Combination of ssDNA recombineering and CRISPR-Cas9 for Pseudomonas putida KT2440 genome editing.
Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon-based plasmid cloned with the tightly regulated redβ and cas9 genes; the second is a broad host range, pBBR1 replicon-based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.
-
Bacterial Retrons Enable Precise Gene Editing in Human Cells.
Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve the efficiency of precise genome editing via homology-directed repair (HDR). In human cells, HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.4%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.
-
Plasmodium falciparum In Vitro Drug Resistance Selections and Gene Editing.
Malaria continues to be a global health burden, threatening over 40% of the world's population. Drug resistance in Plasmodium falciparum, the etiological agent of the majority of human malaria cases, is compromising elimination efforts. New approaches to treating drug-resistant malaria benefit from defining resistance liabilities of known antimalarial agents and compounds in development and defining genetic changes that mediate loss of parasite susceptibility. Here, we present protocols for in vitro selection of drug-resistant parasites and for site-directed gene editing of candidate resistance mediators to test for causality.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.