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Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for...

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Autores principales: Horlbeck, Max A, Gilbert, Luke A, Villalta, Jacqueline E, Adamson, Britt, Pak, Ryan A, Chen, Yuwen, Fields, Alexander P, Park, Chong Yon, Corn, Jacob E, Kampmann, Martin, Weissman, Jonathan S
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5094855/
https://www.ncbi.nlm.nih.gov/pubmed/27661255
http://dx.doi.org/10.7554/eLife.19760
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author Horlbeck, Max A
Gilbert, Luke A
Villalta, Jacqueline E
Adamson, Britt
Pak, Ryan A
Chen, Yuwen
Fields, Alexander P
Park, Chong Yon
Corn, Jacob E
Kampmann, Martin
Weissman, Jonathan S
author_facet Horlbeck, Max A
Gilbert, Luke A
Villalta, Jacqueline E
Adamson, Britt
Pak, Ryan A
Chen, Yuwen
Fields, Alexander P
Park, Chong Yon
Corn, Jacob E
Kampmann, Martin
Weissman, Jonathan S
author_sort Horlbeck, Max A
collection PubMed
description We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.001
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spelling pubmed-50948552016-11-04 Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation Horlbeck, Max A Gilbert, Luke A Villalta, Jacqueline E Adamson, Britt Pak, Ryan A Chen, Yuwen Fields, Alexander P Park, Chong Yon Corn, Jacob E Kampmann, Martin Weissman, Jonathan S eLife Computational and Systems Biology We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.001 eLife Sciences Publications, Ltd 2016-09-23 /pmc/articles/PMC5094855/ /pubmed/27661255 http://dx.doi.org/10.7554/eLife.19760 Text en © 2016, Horlbeck et al http://creativecommons.org/licenses/by/4.0/ This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Computational and Systems Biology
Horlbeck, Max A
Gilbert, Luke A
Villalta, Jacqueline E
Adamson, Britt
Pak, Ryan A
Chen, Yuwen
Fields, Alexander P
Park, Chong Yon
Corn, Jacob E
Kampmann, Martin
Weissman, Jonathan S
Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation
title Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation
title_full Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation
title_fullStr Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation
title_full_unstemmed Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation
title_short Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation
title_sort compact and highly active next-generation libraries for crispr-mediated gene repression and activation
topic Computational and Systems Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5094855/
https://www.ncbi.nlm.nih.gov/pubmed/27661255
http://dx.doi.org/10.7554/eLife.19760
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