Over the past 8 years, the widespread adoption of CRISPR-based technologies has fueled the global genome editing revolution. This platform is based on Cas molecular machines such as Cas9, Cas12, Cas13, as well as other CRISPR effector proteins that are able to alter the genome, transcriptome, and epigenome of virtually any species. Technological improvements have rendered these tools more efficient and precise, and enabled functional diversification and specialization, as recently illustrated by the rise of base editing and the quickly growing demand for prime editing constructs. Here, we discuss the continued adoption of CRISPR tools and constructs distributed by the nonprofit organization Addgene, highlight the trends in the global demand for the CRISPR toolbox, and consider the widespread attitude changes around open sharing that are having a transformative effect on speeding up science.
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http://dx.doi.org/10.1089/crispr.2020.0075 | DOI Listing |
Nucleic Acids Res
January 2025
Beijing Key Laboratory for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing 100083, China.
The flexibility and programmability of CRISPR-Cas technology have made it one of the most popular tools for biomarker diagnostics and gene regulation. Especially, the CRISPR-Cas12 system has shown exceptional clinical diagnosis and gene editing capabilities. Here, we discovered that although the top loop of the 5' handle of guide RNA can undergo central splitting, deactivating CRISPR-Cas12a, the segments can dramatically restore CRISPR function through nucleic acid self-assembly or interactions with small molecules and aptamers.
View Article and Find Full Text PDFNucleic Acids Res
January 2025
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, 620 West Chang'an Avenue, Chang'an District, Xi'an, Shaanxi 710119, P.R. China.
We present a robust 'splice-at-will' CRISPR RNA (crRNA) engineering mechanism that overcomes the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in directly detecting ultrashort RNAs. In this strategy, an intact Cas12a crRNA can be split from almost any site of the spacer region to obtain a truncated crRNA (tcrRNA) that cannot activate Cas12a even after binding an auxiliary DNA activator. While splicing tcrRNAs with a moiety of ultrashort RNA, the formed combination can work together to activate Cas12a efficiently, enabling 'splice-at-will' crRNA engineering.
View Article and Find Full Text PDFSci China Life Sci
January 2025
Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Since their discovery, CRISPR/Cas systems have significantly expanded the genetic toolbox, aiding in the exploration and enhanced production of natural products across various microbes. Among these, class 2 CRISPR/Cas systems are simpler and more broadly used, but they frequently fail to function effectively in many Streptomyces strains. In this study, we present an engineered class 1 type I CRISPR/Cas system derived from Streptomyces avermitilis, which enables efficient gene editing in phylogenetically distant Streptomyces strains.
View Article and Find Full Text PDFBiosci Microbiota Food Health
September 2024
Department of Agricultural Chemistry, Graduate School of Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
Although the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been extensively developed since its discovery for eukaryotic and prokaryotic genome editing and other genetic manipulations, there are still areas warranting improvement, especially regarding bacteria. In this study, BRD0539, a small-molecule inhibitor of Cas9 (SpCas9), was used to suppress the activity of the nuclease during genetic modification of , as well as to regulate CRISPR interference (CRISPRi). First, we developed and validated a CRISPR-SpCas9 system targeting the gene of .
View Article and Find Full Text PDFbioRxiv
December 2024
Key Laboratory of Bioresource Research and Development of Liaoning Province, College of Life and Health Sciences, Northeastern University, Shenyang, 110819, China.
As the toolbox of base editors (BEs) expands, selecting appropriate BE and guide RNA (gRNA) to achieve optimal editing efficiency and outcome for a given target becomes challenging. Here, we construct a set of 10 adenine and cytosine BEs with high activity and broad targeting scope, and comprehensively evaluate their editing profiles and properties head-to-head with 34,040 BE-gRNA-target combinations using genomically integrated long targets and tiling gRNA strategies. Interestingly, we observe widespread non-canonical protospacer adjacent motifs (PAMs) for these BEs.
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