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A customizable protocol for string assembly gRNA cloning (STAgR).

J. Vis. Exp. 142:e58556 (2018)
Publ. Version/Full Text DOI
Open Access Gold (Paid Option)
Creative Commons Lizenzvertrag
The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Genetics ; Issue 142 ; Crispr ; Cas9 ; Dcas9 ; Grna Cloning ; Grna Multiplexing ; Genome Editing ; Transcriptome Editing; One-step Generation; Genome
ISSN (print) / ISBN 1940-087X
e-ISSN 1940-087X
Quellenangaben Volume: 142, Issue: , Pages: , Article Number: e58556 Supplement: ,
Publisher JoVE
Publishing Place 1 Alewife Center, Ste 200, Cambridge, Ma 02140 Usa
Reviewing status Peer reviewed