Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries

Abstract Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target...

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Autores principales:	Swee Hoe Ong, Yilong Li, Hiroko Koike-Yusa, Kosuke Yusa
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/bf61f265b7c34397b4f245df7adcb64e
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id	oai:doaj.org-article:bf61f265b7c34397b4f245df7adcb64e
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spelling	oai:doaj.org-article:bf61f265b7c34397b4f245df7adcb64e2021-12-02T11:41:22ZOptimised metrics for CRISPR-KO screens with second-generation gRNA libraries10.1038/s41598-017-07827-z2045-2322https://doaj.org/article/bf61f265b7c34397b4f245df7adcb64e2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-07827-zhttps://doaj.org/toc/2045-2322Abstract Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target prediction algorithms, have since been made to increase their screening performance. We compared the performance of three available second-generation human genome-wide CRISPR-KO libraries that included at least one of the improvements, and examined the effect of gRNA scaffold, number of gRNAs per gene and number of replicates on screen performance. We identified duplicated screens using a library with 6 gRNAs per gene as providing the best trade-off. Despite the improvements, we found that each improved library still has library-specific false negatives and, for the first time, estimated the false negative rates of CRISPR-KO screens, which are between 10% and 20%. Our newly-defined optimal screening parameters would be helpful in designing screens and constructing bespoke gRNA libraries.Swee Hoe OngYilong LiHiroko Koike-YusaKosuke YusaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Swee Hoe Ong Yilong Li Hiroko Koike-Yusa Kosuke Yusa Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries
description	Abstract Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target prediction algorithms, have since been made to increase their screening performance. We compared the performance of three available second-generation human genome-wide CRISPR-KO libraries that included at least one of the improvements, and examined the effect of gRNA scaffold, number of gRNAs per gene and number of replicates on screen performance. We identified duplicated screens using a library with 6 gRNAs per gene as providing the best trade-off. Despite the improvements, we found that each improved library still has library-specific false negatives and, for the first time, estimated the false negative rates of CRISPR-KO screens, which are between 10% and 20%. Our newly-defined optimal screening parameters would be helpful in designing screens and constructing bespoke gRNA libraries.
format	article
author	Swee Hoe Ong Yilong Li Hiroko Koike-Yusa Kosuke Yusa
author_facet	Swee Hoe Ong Yilong Li Hiroko Koike-Yusa Kosuke Yusa
author_sort	Swee Hoe Ong
title	Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries
title_short	Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries
title_full	Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries
title_fullStr	Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries
title_full_unstemmed	Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries
title_sort	optimised metrics for crispr-ko screens with second-generation grna libraries
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/bf61f265b7c34397b4f245df7adcb64e
work_keys_str_mv	AT sweehoeong optimisedmetricsforcrisprkoscreenswithsecondgenerationgrnalibraries AT yilongli optimisedmetricsforcrisprkoscreenswithsecondgenerationgrnalibraries AT hirokokoikeyusa optimisedmetricsforcrisprkoscreenswithsecondgenerationgrnalibraries AT kosukeyusa optimisedmetricsforcrisprkoscreenswithsecondgenerationgrnalibraries
_version_	1718395424041074688

Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries

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