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CROP: a CRISPR/Cas9 guide selection program based on mapping guide variants.
The off-target effect, in which DNA cleavage was conducted outside the targeted region, is a major problem which limits the applications of CRISPR/Cas9 genome editing system. CRISPR Off-target Predictor (CROP) is standalone program developed to address this problem by predicting off-target propensity of guide RNAs and thereby allowing the user to select the optimum guides. The approach used by CROP involves generating substitution, deletion and insertion combinations which are then mapped into the reference genome. Based on these mapped variants, scoring and alignment are conducted and then reported as a table comprising the off-target propensity of all guide RNAs from a given gene sequence. The Python script for this program is freely available from: https://github.com/vaprilyanto/crop .
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Constructing pyrG marker by CRISPR/Cas facilities the highly-efficient precise genome editing on industrial Aspergillus niger strain.
To prevent the unique difficulty of hygromycin-based gene editing on industrial A. niger strain and increase the working efficiency, the local pyrG marker was removed by well-designed dual sgRNAs and repair template through Cas9-ribonucleoprotein (RNP) strategy in this study. The positive rate of the desired pyrG auxotroph construction was 100%, while no transformant was observed using the traditional methods. The complementation strain showed similar fermentation character as the starting strain. Moreover, an efficient and seamless knock out plasmid-based strategy was established, achieving positive rate at 90% and 50% for challenging Δku70 and Δku80 respectively. Further, combined hygromycin markers and miniaturization cultivation were conducted to select the poor growth strain. Finally, skillfully designed sgRNA and amdS counter-selection repair template were used to obtain ERG3Tyr185 mutation. A highly-efficient precise strategy was established for A. niger through a diagnostic PCR method, with nearly 100% positive rate. Highly- precise desired point mutation was achieved by the developed gene toolbox.
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Transgene-free CRISPR/Cas9-mediated gene editing through protoplast-to-plant regeneration enhances active compounds in Salvia miltiorrhiza.
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Selecting for CRISPR-Edited Knock-In Cells.
CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, "scarless" selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells.
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CRISPR/Cas9-Mediated Editing in FAD2 Gene to Enhance Oil Quality in Soybean [Glycine max (L.) Merrill].
Conventional soybean oil contains high levels of linoleic acid, which reduces oxidative stability and necessitates hydrogenation, leading to trans-fat formation. In this study, 40 Indian soybean genotypes were screened for fatty acid composition, and Gujarat Junagadh Soybean-3 (GJS-3) was selected for CRISPR/Cas9-mediated editing of the fatty acid desaturase-2 (FAD2) gene. Agrobacterium-mediated transformation produced 22 regenerated plants, of which 57.1% were PCR-positive for Cas9/sgRNA. Targeted single-nucleotide substitutions were confirmed by Sanger sequencing in three edited lines (T3, T7, and T15), corresponding to an editing efficiency of 13.63%. These lines exhibited a marked increase in oleic acid content (42-45%) compared with the wild type (22%) and a concomitant reduction in linoleic acid (30-32% vs. 54%), resulting in nearly a two-fold improvement in the oleic/linoleic acid ratio. PCR analysis confirmed the absence of Cas9 and U3 sequences, indicating transgene-free edited plants. This study provides the first evidence of CRISPR/Cas9-mediated FAD2 editing in an Indian soybean cultivar and demonstrates its effectiveness in improving oil quality, oxidative stability, and processing efficiency.
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Manufacturing and Delivering Genome-Editing Proteins.
Genome-editing technologies have revolutionized the biomedical sciences by providing researchers with the ability to quickly and efficiently modify genes. While programmable nucleases can be introduced into cells using a variety of techniques, their delivery as purified proteins is an effective approach for limiting off-target effects. Here, we describe step-by-step procedures for manufacturing and delivering genome-modifying proteins-including Cas9 ribonucleoproteins (RNPs) and TALE and zinc-finger nucleases-into mammalian cells. Protocols for combining Cas9 RNP with naturally recombinogenic adeno-associated virus (AAV) donor vectors for the seamless insertion of transgenes by homology-directed genome editing are also provided.
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Cytosolic delivery of CRISPR/Cas9 ribonucleoproteins for genome editing using chitosan-coated red fluorescent protein.
Though plenty of viral and non-viral methods have been rapidly developed for the delivery of the CRISPR/Cas9 system, the direct delivery of Cas9 ribonucleoproteins (RNPs) into the nucleus for genome editing via the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway, especially the latter one, remains a challenge. Here we report a delivery vehicle achieved by encapsulating red fluorescent protein (RFP) within chitosan (CS), which can simultaneously deliver engineered Cas9 RNPs with a poly-glutamate peptide tag (E-tag) and DNA donors into a range of cell types with high genome editing efficacy and non-cytotoxicity.
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The Vascular Genome as a Therapeutic Target: A Systematic Review of CRISPR-based Gene Editing In Vascular Disease.
Despite advances in therapy, arterial, venous, and pulmonary vascular diseases remain leading causes of morbidity and mortality. Persistent endothelial dysfunction, inflammation, oxidative stress, and maladaptive vascular remodeling continue to drive disease progression and residual risk. CRISPR/Cas9 technology offers a unique opportunity to modify the molecular pathways underlying vascular pathophysiology directly. The PRISMA 2020 guidelines guided the systematic review. The databases PubMed/MEDLINE, Embase, Web of Science, Cochrane Library, ClinicalTrials.gov, and Google Scholar were searched from their inception until September 2025 for experimental and/or clinical studies evaluating the application of CRISPR/Cas9 on vascular disease. Included were in vitro studies, animal model studies, and early-phase human studies aimed at targeting the endothelial cell regulatory pathways, inflammatory pathways, metabolic remodeling processes, and hereditary causes of vasculopathy. Seventeen studies met the inclusion criteria. CRISPR technologies targeting PCSK9, NOS3, HIF1A, NLRP3, METTL4, BMPR2, and ACTA2 were identified to enhance repair mechanisms in endothelial cells, regulate inflammation, modulate lipid metabolism, and remodel the vascular system. The human studies demonstrated sustained gene silencing effects following a single dose of CRISPR-induced in vivo editing. The use of CRISPR technology to edit cell genomes offers potential to alter disease progression in vascular medicine, with a growing body of translational evidence supporting the feasibility and durability of the approach.
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Highly Efficient CRISPR-Associated Protein 9 Ribonucleoprotein-Based Genome Editing in Euglena gracilis.
Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, and biofuels. However, targeted mutagenesis in this species has been a long-standing challenge. We recently developed a transgene-free, highly efficient, genome editing method for E. gracilis using CRISPR/Cas9 ribonucleoproteins (RNPs). Our method achieved mutagenesis rates of approximately 80% or more through an electroporation-based direct delivery of Cas9 RNPs. Therefore, this method is suitable for basic research and industrial applications, such as the breeding of Euglena. For complete details on the use and execution of this protocol, please refer to Nomura et al. (2019).
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Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects.
Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.
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