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Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

BACKGROUND: In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding...

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Autores principales: Henkel, Luisa, Rauscher, Benedikt, Schmitt, Barbara, Winter, Jan, Boutros, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686728/
https://www.ncbi.nlm.nih.gov/pubmed/33228647
http://dx.doi.org/10.1186/s12915-020-00905-1
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author Henkel, Luisa
Rauscher, Benedikt
Schmitt, Barbara
Winter, Jan
Boutros, Michael
author_facet Henkel, Luisa
Rauscher, Benedikt
Schmitt, Barbara
Winter, Jan
Boutros, Michael
author_sort Henkel, Luisa
collection PubMed
description BACKGROUND: In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding and noncoding genome of numerous cell types in different organisms have been interrogated. However, screening designs vary greatly and criteria for optimal experimental implementation and library composition are still emerging. Given their broad utility in functionally annotating genomes, the application and interpretation of genome-scale CRISPR screens would greatly benefit from consistent and optimal design criteria. RESULTS: We report advantages of conducting viability screens in selected Cas9 single-cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg) RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a novel genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. CONCLUSION: We show how empirically designed libraries in combination with an optimized experimental design increase the dynamic range in gene essentiality screens at reduced library coverage. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-020-00905-1.
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spelling pubmed-76867282020-11-25 Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library Henkel, Luisa Rauscher, Benedikt Schmitt, Barbara Winter, Jan Boutros, Michael BMC Biol Methodology Article BACKGROUND: In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding and noncoding genome of numerous cell types in different organisms have been interrogated. However, screening designs vary greatly and criteria for optimal experimental implementation and library composition are still emerging. Given their broad utility in functionally annotating genomes, the application and interpretation of genome-scale CRISPR screens would greatly benefit from consistent and optimal design criteria. RESULTS: We report advantages of conducting viability screens in selected Cas9 single-cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg) RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a novel genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. CONCLUSION: We show how empirically designed libraries in combination with an optimized experimental design increase the dynamic range in gene essentiality screens at reduced library coverage. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-020-00905-1. BioMed Central 2020-11-23 /pmc/articles/PMC7686728/ /pubmed/33228647 http://dx.doi.org/10.1186/s12915-020-00905-1 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data.
spellingShingle Methodology Article
Henkel, Luisa
Rauscher, Benedikt
Schmitt, Barbara
Winter, Jan
Boutros, Michael
Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
title Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
title_full Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
title_fullStr Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
title_full_unstemmed Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
title_short Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
title_sort genome-scale crispr screening at high sensitivity with an empirically designed sgrna library
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686728/
https://www.ncbi.nlm.nih.gov/pubmed/33228647
http://dx.doi.org/10.1186/s12915-020-00905-1
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