1. Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome
- Author
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Rahel de Bruyn, Simone Schaubeck, Valentina Diehl, Michael G.B. Hayes, Ivan Dikic, Yves Matthess, Marie Hebel, Svenja Wiechmann, Sven Heinz, Verena Bittl, Christopher Benner, Andreas Ernst, Martin Wegner, Anja Bremm, Manuel Kaulich, Publica, and Weissman, Jonathan
- Subjects
0301 basic medicine ,Genome ,Deubiquitinating enzyme ,genome-wide ,0302 clinical medicine ,cell biology ,CRISPR ,genetics ,Clustered Regularly Interspaced Short Palindromic Repeats ,Guide RNA ,Kinetoplastida ,Biology (General) ,biology ,General Neuroscience ,General Medicine ,Tools and Resources ,030220 oncology & carcinogenesis ,Gene Targeting ,Medicine ,gRNA library ,DUBs ,Biotechnology ,RNA, Guide, Kinetoplastida ,Human ,QH301-705.5 ,Science ,3Cs technology ,Genomics ,Computational biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,03 medical and health sciences ,CRISPR/Cas ,genomics ,Humans ,ddc:610 ,human ,Transcription factor ,Gene ,General Immunology and Microbiology ,Human Genome ,Genetics and Genomics ,Cell Biology ,Endonucleases ,Quality Education ,030104 developmental biology ,Mutagenesis ,Doxorubicin ,biology.protein ,RNA ,Human genome ,Biochemistry and Cell Biology ,Guide - Abstract
Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
- Published
- 2019