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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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2016
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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 |
format | Online Article Text |
id | pubmed-5094855 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
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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