CRISPR-based genome editing has been widely implemented in various cell types. In silico single guide RNA (sgRNA) design is a key step for successful gene editing using the CRISPR system, and continuing efforts are aimed at refining in silico sgRNA design with high on-target efficacy and reduced off-target effects. Many sgRNA design tools are available, but careful assessments of their application scenarios and performance benchmarks across different types of genome-editing data are needed. Efficient in silico models can be built that integrate current heterogeneous genome-editing data to derive unbiased sgRNA design rules and identify key features for improving sgRNA design. Comprehensive evaluation of on-target and off-target effects of sgRNA will allow more precise genome editing and gene therapies using the CRISPR system.
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http://dx.doi.org/10.1016/j.tibtech.2016.06.008 | DOI Listing |
Int J Biol Macromol
December 2024
National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, Hubei 430070, China; Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture of the People's Republic of China, Wuhan, Hubei 430070, China; International Research Center for Animal Disease, Ministry of Science and Technology of the People's Republic of China, Wuhan, Hubei 430070, China; Guangdong Provincial Key Laboratory of Research on the Technology of Pig-breeding and Pig-disease prevention, Guangzhou, Guangdong 510000, China. Electronic address:
The avian influenza virus (AIV) poses a significant threat to both the poultry industry and public health. Systematic identification of host factors involved in AIV infection in chicken is critical. In this study, we developed a comprehensive chicken genome-wide sgRNA library containing 76,350 sgRNAs, with 4-6 sgRNAs designed per gene.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
Sanya Institute of Nanjing Agricultural University, Department of Plant Pathology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
The establishment of reliable and efficient systems for genome editing in Phytophthora is very important for studying gene functions. Here, step-by-step methods for CRISPR/Cas9-based gene knockout and in situ complementation for Phytophthora sojae are presented. These steps include the sgRNA design, Cas9-sgRNA plasmid construction, homologous replacement, complementation vector construction, P.
View Article and Find Full Text PDFGenome Med
December 2024
Department of Biomedical Engineering, Tel Aviv University, Tel-Aviv, 6997801, Israel.
Background: CRISPR is widely used to silence genes by inducing mutations expected to nullify their expression. While numerous computational tools have been developed to design single-guide RNAs (sgRNAs) with high cutting efficiency and minimal off-target effects, only a few tools focus specifically on predicting gene knockouts following CRISPR. These tools consider factors like conservation, amino acid composition, and frameshift likelihood.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
December 2024
Institute of Molecular Medicine, Renji Hospital, School of Medicine Shanghai Jiao Tong University, Shanghai 200127, China.
Artificially functional RNAs, such as fluorogenic RNA aptamer (FRApt)-based biosensing tag, represent significant advancements in various biological applications but are limited by the lack of insight into dynamic structure ensembles and universal design concepts. Through the development of an artificial RNA structure ensemble, we rationally established an RNA reconstitution model, "SSPepper-Apt," to generate a universal fluorogenic RNA biosensing tag. By utilizing various target-recognizing RNA motifs, SSPepper-Apt enables the modular generation of sensing tags for low-background, highly selective imaging of metabolites, peptides, and proteins in living cells.
View Article and Find Full Text PDFSTAR Protoc
December 2024
Institute Pharmaceutical Biology/DCAL, Goethe-University, 60438 Frankfurt am Main, Germany. Electronic address:
KMT2A rearrangements are associated with a poor clinical outcome in infant, pediatric, and adult acute lymphoblastic and myeloid leukemia. Here, we present a protocol to reconstruct chromosomal translocations with different partner genes of KMT2A in vitro. We describe steps for patient-specific single guide RNA (sgRNA) design, optimized sgRNA in vitro transcription, detailed purification of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB), and CRISPR-Cas9 editing of the test cell line K562 as well as UCB HSPCs.
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