The CRISPR-Cas9 system has recently evolved as a powerful mutagenic tool for targeted genome editing. The impeccable functioning of the system depends on the optimal design of single guide RNAs (sgRNAs) that mainly involves sgRNA specificity and on-target cleavage efficacy. Several research groups have designed algorithms and models, trained on mammalian genomes, for predicting sgRNAs cleavage efficacy. These models are also implemented in most plant sgRNA design tools due to the lack of on-target cleavage efficacy studies in plants. However, one of the major drawbacks is that almost all of these models are biased for considering only coding regions of the DNA while excluding ineffective regions, which are of immense importance in functional genomics studies especially for plants, thus making prediction less reliable. In the present study, we evaluate the on-target cleavage efficacy of experimentally validated sgRNAs designed against diverse ineffective regions of genome using various statistical tests. We show that nucleotide preference in protospacer adjacent motif (PAM) proximal region, GC content in the PAM proximal seed region, intact RAR and 3 stem loop structures, and free accessibility of nucleotides in seed and tracrRNA regions of sgRNAs are important determinants associated with their high on-target cleavage efficacy. Thus, our study describes the features important for plant sgRNAs high on-target cleavage efficacy against ineffective genomic regions previously shown to give rise to ineffective sgRNAs. Moreover, it suggests the need of developing an elaborative plant-specific sgRNA design model considering the entire genomic landscape including ineffective regions for enabling highly efficient genome editing without wasting time and experimental resources.
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http://dx.doi.org/10.7717/peerj.11409 | DOI Listing |
J Agric Food Chem
January 2025
Faculty of Mechanical and Process Engineering, Hochschule Offenburg, 77652 Offenburg, Germany.
Protein hydrolysis under acidic conditions can improve the product quality, nutrient availability, and cost efficiency, particularly when neutral or alkaline enzymes are ineffective. Six fungal aspartic endopeptidases (FAPs) were recombinantly expressed as active enzymes in , with peak activity between 30-50 °C and pH 3.0-4.
View Article and Find Full Text PDFBr J Cancer
January 2025
Department of Liver Surgery, Sun Yat-sen University Cancer Center, Guangzhou, China.
Background: Pyroptosis is closely associated with chemotherapeutic drugs and immune response. Here, we investigated whether oxaliplatin, a key drug in FOLFOX-hepatic artery infusion chemotherapy (FOLFOX-HAIC), induces pyroptosis in hepatoma cells and enhances antitumor immunity after tumor cell death.
Methods: Hepatoma cells were treated with oxaliplatin.
Bioelectrochemistry
December 2024
Department of Laboratory Medicine, The Affiliated Hospital of Southwest Medical University, 25 Taiping Street, Luzhou 646000, Sichuan, China; Sichuan Province Engineering Technology Research Center of Molecular Diagnosis of Clinical Diseases, China; Molecular Diagnosis of Clinical Diseases Key Laboratory of Luzhou, Sichuan, China. Electronic address:
In this study, an innovative electrochemical biosensor was developed for the rapid, specific, and sensitive detection of Acinetobacter baumannii without the need for sample pretreatment. The biosensor utilized an aptamer as a specific capture probe for A. baumannii and employed a self-powered DNAzyme walker cleavage cycle reaction to achieve signal amplification.
View Article and Find Full Text PDFJ Colloid Interface Sci
December 2024
Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China; Joint International Research Laboratory of Intelligent Drug Delivery Systems, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China. Electronic address:
Optimizing the design of nanoparticulate co-delivery systems of antigens and immunomodulators to induce antigen-specific immune tolerance effectively remains a challenge, constrained by low drug loading capacity and premature leakage of active ingredients. Here, we report a prodrug self-assembled nanoparticles (NPs) strategy to synergistically deliver antigen and rapamycin (RAPA) into antigen-presenting cells (APCs) by simply conjugating rapamycin with an aliphatic chain. These prodrug NPs can be efficiently taken up by APCs and then release rapamycin through cleavage of the linker by intracellular esterase.
View Article and Find Full Text PDFNat Commun
December 2024
Beijing Frontier Research Center for Biological Structure, State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Exceptionally diverse type V CRISPR-Cas systems provide numerous RNA-guided nucleases as powerful tools for DNA manipulation. Two known Cas12e nucleases, DpbCas12e and PlmCas12e, are both effective in genome editing. However, many differences exist in their in vitro dsDNA cleavage activities, reflecting the diversity in Cas12e's enzymatic properties.
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