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: 26 190
Страница 575 из 1000-
Cas12h is a crRNA-guided DNA nickase that can be utilized for precise gene editing.
Type V-H CRISPR-Cas system, an important subtype of type V CRISPR-Cas systems, has remained enigmatic in terms of its structure and function despite being discovered several years ago. Here, we comprehensively characterize the type V-H CRISPR-Cas system and elucidate its role as a DNA nicking system. The unique CRISPR RNA (crRNA) employed by Cas12h effector protein enables specific targeting of double-stranded DNA (dsDNA), while its RuvC domain is responsible for cleaving the non-target strand (NTS) of dsDNA. We present the structure of Cas12h bound to crRNA and target DNA. Our structural analysis reveals that the RuvC domain possesses a narrow active pocket that facilitates recognition of NTS but potentially hinders access to the target strand. Furthermore, we demonstrate that Cas12h confers adaptive immunity against invading mobile genetic elements through transcriptional gene inhibition. We have engineered an adenine base editor by fusing Cas12h with an adenine deaminase, achieving effective A-to-G substitution.
-
Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit.
Genome editing facilitated by Cas9-based RNA-guided nucleases (RGNs) is becoming an increasingly important and popular technique for reverse genetics in both model and non-model species. So far, RGNs were mainly applied for the induction of point mutations, and one major challenge consists in the detection of genome-edited individuals from a mutagenized population. Also, point mutations are not appropriate for functional dissection of non-coding DNA. Here, the multiplexing capacity of a newly developed genome editing toolkit was exploited for the induction of inheritable chromosomal deletions at six different loci in Nicotiana benthamiana and Arabidopsis. In both species, the preferential formation of small deletions was observed, suggesting reduced efficiency with increasing deletion size. Importantly, small deletions (<100 bp) were detected at high frequencies in N. benthamiana T0 and Arabidopsis T2 populations. Thus, targeting of small deletions by paired nucleases represents a simple approach for the generation of mutant alleles segregating as size polymorphisms in subsequent generations. Phenotypically selected deletions of up to 120 kb occurred at low frequencies in Arabidopsis, suggesting larger population sizes for the discovery of valuable alleles from addressing gene clusters or non-coding DNA for deletion by programmable nucleases.
-
CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice.
-
Rock paper scissors: CRISPR/Cas9-mediated interference with geminiviruses in plants.
-
Revolutionizing Tuberculosis Management With Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas Technology: A Comprehensive Literature Review.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have gained attention for their revolutionary potential in tuberculosis (TB) management, providing a novel approach to both diagnostics and treatment. This technology, renowned for its ability to accurately target and modify genetic material, offers a promising solution to the limitations of current TB diagnostic methods, which often rely on time-consuming culture techniques or polymerase chain reaction (PCR)-based assays. One of the key advantages of CRISPR-Cas systems is their high specificity and sensitivity, making them well-suited for detecting Mycobacterium tuberculosis, even in low-bacterial-load samples. Techniques such as CRISPR-Cas12 and Cas13 have been employed for rapid detection, utilizing their trans-cleavage activity to produce a fluorescent signal upon recognition of the TB genome. Furthermore, these methods often use isothermal amplification techniques like recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), which require less equipment compared to traditional PCR. Beyond diagnostics, CRISPR-Cas technologies show promise in studying TB resistance mechanisms and potentially treating drug-resistant strains. Genome-editing capabilities enable researchers to manipulate the M. tuberculosis genome, investigating genes linked to virulence or antibiotic resistance. Although challenges such as the development of multiplexed CRISPR assays for detecting multiple mutations simultaneously remain, advancements continue to improve the technology's practicality for clinical use. Incorporating CRISPR into TB management could enhance early detection, inform personalized treatment, and potentially contribute to developing more effective therapies, especially in regions where TB remains a significant public health threat.
-
Application of CRISPR-Cas9 gene editing for congenital heart disease.
Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is an ancient prokaryotic defense system that precisely cuts foreign genomic DNA under the control of a small number of guide RNAs. The CRISPR-Cas9 system facilitates efficient double-stranded DNA cleavage that has been recently adopted for genome editing to create or correct inherited genetic mutations causing disease. Congenital heart disease (CHD) is generally caused by genetic mutations such as base substitutions, deletions, and insertions, which result in diverse developmental defects and remains a leading cause of birth defects. Pediatric CHD patients exhibit a spectrum of cardiac abnormalities such as septal defects, valvular defects, and abnormal chamber development. CHD onset occurs during the prenatal period and often results in early lethality during childhood. Because CRISPR-Cas9-based genome editing technology has gained considerable attention for its potential to prevent and treat diseases, we will review the CRISPR-Cas9 system as a genome editing tool and focus on its therapeutic application for CHD.
-
CRISPR/Cas9-mediated knockin of IRES-tdTomato at Ins2 locus reveals no RFP-positive cells in mouse islets.
Using the CRISPR/Cas9 genomic editing technology, we constructed a transgenic mouse model to express specific fluorescent protein in pancreatic β cells, which harbor tdTomato exogenous gene downstream of the Ins2 promoter in C57BL/6 J mice. The Ins2-specific single-guide RNA-targeted exon2 was designed for the CRISPR/Cas9 system and Donor vector was constructed at the same time. Then Cas9, sgRNA, and Donor vector were microinjected in vitro into the mouse zygotes that were implanted into pseudo-pregnant mice. We obtained homozygotes through mating heterozygotes, and verified the knockin effect through genotype identification, in vivo imaging, and frozen section. Six F0 mice and stable inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed that the knockin group had no change in the Ins2 exon compared with the control group, while only the base sequence of tdTomato was added and no base mutation occurred. However, in vivo imaging and frozen section did not observe the expression of red fluorescent protein (RFP), and the protein expression of knockin gene tdTomato was negative. As a result, the expressions of tdTomato protein and fluorescence intensity were low and the detection threshold was not reached. In the CRISP/Cas9 technique, the exogenous fragment of IRES connection would affect the transcription level of the preceding gene, which in turn would lead to low-level expression of the downstream gene and affect the effect of gene insertion.
-
Prime editing outperforms homology-directed repair as a tool for CRISPR-mediated variant knock-in in zebrafish.
Zebrafish serve as a valuable model organism for studying human genetic diseases. While generating knockout lines is relatively straightforward, introducing precise disease-specific genetic variants by knock-in (KI) remains challenging. KI lines, however, enable more accurate studies of molecular and physiological consequences of genetic diseases. Their generation is often hampered by low editing efficiency (EE) and potential off-target effects. Here, we optimized conventional CRISPR-Cas9-mediated homology-directed repair (HDR) strategies for precise KI of genetic variants in zebrafish and compared their efficacy with prime editing, a recently developed technique that is not yet commonly used. Using next-generation sequencing, we determined KI EE by HDR for six unique base-pair substitutions in three different zebrafish genes. We assessed the effect of (1) varying Cas9 amounts, (2) HDR templates with chemical modifications to improve integration efficiency, (3) different microinjection procedures and (4) introduction of additional synonymous guide-blocking variants in the HDR template. Increasing Cas9 amounts augmented KI EE, with optimal injected amounts of Cas9 between 200 pg and 800 pg. The use of Alt-R HDR templates further increased KI EE, while guide-blocking modifications did not. Injecting components directly into the cell was not superior to injections into the yolk. Prime editing, however, increased EE up to fourfold and expanded the F0 founder pool for four targets compared with conventional HDR editing, with fewer off-target effects. Therefore, prime editing is a very promising methodology for improving the creation of precise genomic edits in zebrafish, facilitating the modeling of human diseases.
-
Rapid customization of base editors via machine learning-powered combinatorial mutagenesis.
Being able to control the complementarity and hindrance between target DNA and base editor proteins enables precise, bystander-free editing. Here, we combined combinatorial mutagenesis with machine learning to analyze and engineer these interactions at scale. By profiling DNA motif preferences across 160,000 evoAPOBEC1 and 64 million TadA variants in human cells, we used as little as 0.004% of the mutational landscape to make predictions. This identified variants with motif-specific activity and eliminated residual adenine editing in cytosine base editors. In correcting >800 disease-associated mutations, our variants outperformed previous versions in precluding unintended edits at purine motifs, achieving undetectable bystander edits in 50% of cases. Additionally, a pre-trained, structure-based deep learning model predicted functional TadA variants with 63% success across 2026 variants spanning 26 amino acid sites, without experimental data and in a single prediction round. These approaches streamline the re-engineering of base editors for enhanced precision tailored to specific targets.
-
A modified glycosylase base editor without predictable DNA off-target effects.
Glycosylase base editor (GBE) can induce C-to-G transversion in mammalian cells, showing great promise for the treatment of human genetic disorders. However, the limited efficiency of transversion and the possibility of off-target effects caused by Cas9 restrict its potential clinical applications. In our recent study, we have successfully developed TaC9-CBE and TaC9-ABE by separating nCas9 and deaminase, which eliminates the Cas9-dependent DNA off-target effects without compromising editing efficiency. We developed a novel GBE called TaC9-GBEYE1, which utilizes the deaminase and UNG-nCas9 guided by TALE and sgRNA, respectively. TaC9-GBEYE1 showed comparable levels of on-target editing efficiency to traditional GBE at 19 target sites, without any off-target effects caused by Cas9 or TALE. The TaC9-GBEYE1 is a safe tool for gene therapy.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.