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Köferle, A.* ; Stricker, S.H.

A universal protocol for large-scale gRNA library production from any DNA source.

J. Vis. Exp. 2017:e56264 (2017)
Verlagsversion DOI PMC
Free by publisher
Creative Commons Lizenzvertrag
Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
Schlagwörter Genetics ; Issue 130 ; Grna Library ; Crispr ; Cas9 ; Nuclease ; Library Generation ; Genome-wide; Human-cells
ISSN (print) / ISBN 1940-087X
e-ISSN 1940-087X
Zeitschrift Journal of Visualized Experiments
Quellenangaben Band: 2017, Heft: 130, Seiten: , Artikelnummer: e56264 Supplement: ,
Verlag JoVE
Verlagsort Cambridge
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Stem Cell Research (ISF)