Your browser does not allow saving data (e.g. private mode): bookmarks will be lost when the tab closes.
Search PubMed
Keywords, author, journal, years — results from PubMed, abstracts with Russian translation, bookmarks in your browser.
Found: 26 147
Page 494 of 1000-
Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e.
CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.
-
A transient reporter for editing enrichment (TREE) in human cells.
Current approaches to identify cell populations that have been modified with deaminase base editing technologies are inefficient and rely on downstream sequencing techniques. In this study, we utilized a blue fluorescent protein (BFP) that converts to green fluorescent protein (GFP) upon a C-to-T substitution as an assay to report directly on base editing activity within a cell. Using this assay, we optimize various base editing transfection parameters and delivery strategies. Moreover, we utilize this assay in conjunction with flow cytometry to develop a transient reporter for editing enrichment (TREE) to efficiently purify base-edited cell populations. Compared to conventional cell enrichment strategies that employ reporters of transfection (RoT), TREE significantly improved the editing efficiency at multiple independent loci, with efficiencies approaching 80%. We also employed the BFP-to-GFP conversion assay to optimize base editor vector design in human pluripotent stem cells (hPSCs), a cell type that is resistant to genome editing and in which modification via base editors has not been previously reported. At last, using these optimized vectors in the context of TREE allowed for the highly efficient editing of hPSCs. We envision TREE as a readily adoptable method to facilitate base editing applications in synthetic biology, disease modeling, and regenerative medicine.
-
DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing.
Expanding mitochondrial base editing tools with broad sequence compatibility is of high need for both research and therapeutic purposes. In this study, we identify a DddA homolog from Simiaoa sunii (Ddd_Ss) which can efficiently deaminate cytosine in DC context in double-stranded DNA (dsDNA). We successfully develop Ddd_Ss-derived cytosine base editors (DdCBE_Ss) and introduce mutations at multiple mitochondrial DNA (mtDNA) loci including disease-associated mtDNA mutations in previously inaccessible GC context. Finally, by introducing a single amino acid substitution from Ddd_Ss, we successfully improve the activity and sequence compatibility of DdCBE derived from DddA of Burkholderia cenocepacia (DdCBE_Bc). Our study expands mtDNA editing tool boxes and provides resources for further screening and engineering dsDNA base editors for biological and therapeutic applications.
-
Inheritance of co-edited genes by CRISPR-based targeted nucleotide substitutions in rice.
The CRISPR/Cas9 system is a revolutionary genome-editing tool for directed gene editing in various organisms. Cas9 variants can be applied as molecular homing devices when combined with various functional effectors such as transcriptional activators or DNA modification enzymes. Target-AID is a synthetic complex of nuclease deficient Cas9 fused to an activation-induced cytidine deaminase (AID) that enables targeted nucleotide substitution (C to T or G to A). We previously demonstrated that the introduction of desired point mutations into target genes by Target-AID confers herbicide tolerance to rice callus. Inheritance of the introduced mutations, as well as the removal of transgenes, are key issues that must be addressed in order to fully develop Target-AID as a plant breeding technique. Here we report the transmission of such mutations from the callus to regenerants and their progenies, leading to a generation of selectable marker-free (SMF) herbicide tolerant rice plants with simultaneous multiplex nucleotide substitutions. These findings demonstrate that Target-AID can be developed into novel plant breeding technology which enables improvement of multiplex traits at one time in combination with sophisticated targeted base editing with the simplicity and versatility of CRISPR/Cas9 system.
-
AI-guided Cas9 engineering provides an effective strategy to enhance base editing.
Precise genome editing is crucial for functional studies and therapies. Base editors, while powerful, require optimization for efficiency. Meanwhile, emerging protein design methods and protein language models have driven efficient and intelligent protein engineering. In this study, we employed the Protein Mutational Effect Predictor (ProMEP) to predict the effects of single-site saturated mutations in Cas9 protein, using AncBE4max as the prototype to construct and test 18 candidate point mutations. Based on this, we further predicted combinations of multiple mutations and successfully developed a high-performance variant AncBE4max-AI-8.3, achieving a 2-3-fold increase in average editing efficiency. Introducing the engineered Cas9 into CGBE, YEE-BE4max, ABE-max, and ABE-8e improved their editing performance. The same strategy also substantially improves the efficiencies of HF-BEs. Stable enhancement in editing efficiency was also observed across seven cancer cell lines and human embryonic stem cells. In conclusion, we validated that AI models can serve as more effective protein engineering tools, providing a universal improvement strategy for a series of gene editing tools.
-
Targeted A-to-G base editing of chloroplast DNA in plants.
Chloroplast DNA (cpDNA) encodes up to 315 (typically, 120-130) genes1, including those for essential components in photosystems I and II and the large subunit of RuBisCo, which catalyses CO2 fixation in plants. Targeted mutagenesis in cpDNA will be broadly useful for studying the functions of these genes in molecular detail and for developing crops and other plants with desired traits. Unfortunately, CRISPR-Cas9 and CRISPR-derived base editors, which enable targeted genetic modifications in nuclear DNA, are not suitable for organellar DNA editing2, owing to the difficulty of delivering guide RNA into organelles. CRISPR-free, protein-only base editors (including DddA-derived cytosine base editors3-8 and zinc finger deaminases9), originally developed for mitochondrial DNA editing in mammalian cells, can be used for C-to-T, rather than A-to-G, editing in cpDNA10-12. Here we show that heritable homoplasmic A-to-G edits can be induced in cpDNA, leading to phenotypic changes, using transcription activator-like effector-linked deaminases13.
-
Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox.
CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.
-
Blood vessel organoids generated by base editing and harboring single nucleotide variation in Notch3 effectively recapitulate CADASIL-related pathogenesis.
Human blood vessel organoids (hBVOs) offer a promising platform for investigating vascular diseases and identifying therapeutic targets. In this study, we focused on in vitro modeling and therapeutic target finding of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary stroke disorder caused by mutations in the NOTCH3 gene. Despite the identification of these mutations, the underlying pathological mechanism is elusive, and effective therapeutic approaches are lacking. CADASIL primarily affects the blood vessels in the brain, leading to ischemic strokes, migraines, and dementia. By employing CRISPR/Cas9 base-editing technology, we generated human induced pluripotent stem cells (hiPSCs) carrying Notch3 mutations. These mutant hiPSCs were differentiated into hBVOs. The NOTCH3 mutated hBVOs exhibited CADASIL-like pathology, characterized by a reduced vessel diameter and degeneration of mural cells. Furthermore, we observed an accumulation of Notch3 extracellular domain (Notch3ECD), increased apoptosis, and cytoskeletal alterations in the NOTCH3 mutant hBVOs. Notably, treatment with ROCK inhibitors partially restored the disconnection between endothelial cells and mural cells in the mutant hBVOs. These findings shed light on the pathogenesis of CADASIL and highlight the potential of hBVOs for studying and developing therapeutic interventions for this debilitating human vascular disorder.
-
UNCOVERseq enables sensitive and controlled gene editing off-target nomination across CRISPR-Cas modalities and systems.
The rapid expansion of CRISPR-Cas gene editing enables new therapeutic strategies but complicates assessment of unintended editing risks due to emerging modalities and unclear analytical standards. We present UNCOVERseq (Unbiased Nomination of CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow that sensitively identifies rare off-target events using defined inputs and analytical process controls. Using an inter-method off-target confirmation benchmarking dataset, UNCOVERseq demonstrates high analytical sensitivity (97.6%) and precision (78%), outperforming published nomination methods. We apply UNCOVERseq across 192 guide RNAs and identify six guides spanning a broad specificity range, enabling relative risk assessment across S. pyogenes Cas9, high-fidelity variants, and base editors in hematopoietic stem and progenitor cells. We further show that double-strand break nomination sites retain strong rank-order concordance with single-strand break-mediated base editing. Together, these results establish UNCOVERseq as a robust framework for informed off-target risk assessment in translational gene-editing systems.
-
Editing microbes to mitigate enteric methane emissions in livestock.
Livestock production significantly contributes to greenhouse gas (GHG) emissions particularly methane (CH4) emissions thereby influencing climate change. To address this issue further, it is crucial to establish strategies that simultaneously increase ruminant productivity while minimizing GHG emissions, particularly from cattle, sheep, and goats. Recent advancements have revealed the potential for modulating the rumen microbial ecosystem through genetic selection to reduce methane (CH4) production, and by microbial genome editing including CRISPR/Cas9, TALENs (Transcription Activator-Like Effector Nucleases), ZFNs (Zinc Finger Nucleases), RNA interference (RNAi), Pime editing, Base editing and double-stranded break-free (DSB-free). These technologies enable precise genetic modifications, offering opportunities to enhance traits that reduce environmental impact and optimize metabolic pathways. Additionally, various nutrition-related measures have shown promise in mitigating methane emissions to varying extents. This review aims to present a future-oriented viewpoint on reducing methane emissions from ruminants by leveraging CRISPR/Cas9 technology to engineer the microbial consortia within the rumen. The ultimate objective is to develop sustainable livestock production methods that effectively decrease methane emissions, while maintaining animal health and productivity.
PubMed returns only the first 10,000 results — refine the query to see the rest.