Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements

To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a functio...

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Autores principales: Karvelis, Tautvydas, Gasiunas, Giedrius, Young, Joshua, Bigelyte, Greta, Silanskas, Arunas, Cigan, Mark, Siksnys, Virginijus
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Method
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653880/
https://www.ncbi.nlm.nih.gov/pubmed/26585795
http://dx.doi.org/10.1186/s13059-015-0818-7
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author Karvelis, Tautvydas
Gasiunas, Giedrius
Young, Joshua
Bigelyte, Greta
Silanskas, Arunas
Cigan, Mark
Siksnys, Virginijus
author_facet Karvelis, Tautvydas
Gasiunas, Giedrius
Young, Joshua
Bigelyte, Greta
Silanskas, Arunas
Cigan, Mark
Siksnys, Virginijus
author_sort Karvelis, Tautvydas
collection PubMed
description To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13059-015-0818-7) contains supplementary material, which is available to authorized users.
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spelling pubmed-46538802015-11-21 Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements Karvelis, Tautvydas Gasiunas, Giedrius Young, Joshua Bigelyte, Greta Silanskas, Arunas Cigan, Mark Siksnys, Virginijus Genome Biol Method To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13059-015-0818-7) contains supplementary material, which is available to authorized users. BioMed Central 2015-11-19 2015 /pmc/articles/PMC4653880/ /pubmed/26585795 http://dx.doi.org/10.1186/s13059-015-0818-7 Text en © Karvelis et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Method
Karvelis, Tautvydas
Gasiunas, Giedrius
Young, Joshua
Bigelyte, Greta
Silanskas, Arunas
Cigan, Mark
Siksnys, Virginijus
Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements
title Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements
title_full Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements
title_fullStr Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements
title_full_unstemmed Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements
title_short Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements
title_sort rapid characterization of crispr-cas9 protospacer adjacent motif sequence elements
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653880/
https://www.ncbi.nlm.nih.gov/pubmed/26585795
http://dx.doi.org/10.1186/s13059-015-0818-7
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