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acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens

High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides...

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Autores principales: Ramesh, Adithya, Trivedi, Varun, Lee, Sangcheon, Tafrishi, Aida, Schwartz, Cory, Mohseni, Amirsadra, Li, Mengwan, Lonardi, Stefano, Wheeldon, Ian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10250353/
https://www.ncbi.nlm.nih.gov/pubmed/37291233
http://dx.doi.org/10.1038/s42003-023-04996-8
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author Ramesh, Adithya
Trivedi, Varun
Lee, Sangcheon
Tafrishi, Aida
Schwartz, Cory
Mohseni, Amirsadra
Li, Mengwan
Lonardi, Stefano
Wheeldon, Ian
author_facet Ramesh, Adithya
Trivedi, Varun
Lee, Sangcheon
Tafrishi, Aida
Schwartz, Cory
Mohseni, Amirsadra
Li, Mengwan
Lonardi, Stefano
Wheeldon, Ian
author_sort Ramesh, Adithya
collection PubMed
description High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica and acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.
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spelling pubmed-102503532023-06-10 acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens Ramesh, Adithya Trivedi, Varun Lee, Sangcheon Tafrishi, Aida Schwartz, Cory Mohseni, Amirsadra Li, Mengwan Lonardi, Stefano Wheeldon, Ian Commun Biol Article High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica and acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest. Nature Publishing Group UK 2023-06-08 /pmc/articles/PMC10250353/ /pubmed/37291233 http://dx.doi.org/10.1038/s42003-023-04996-8 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Ramesh, Adithya
Trivedi, Varun
Lee, Sangcheon
Tafrishi, Aida
Schwartz, Cory
Mohseni, Amirsadra
Li, Mengwan
Lonardi, Stefano
Wheeldon, Ian
acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens
title acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens
title_full acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens
title_fullStr acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens
title_full_unstemmed acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens
title_short acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens
title_sort accrispr: an activity-correction method for improving the accuracy of crispr screens
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10250353/
https://www.ncbi.nlm.nih.gov/pubmed/37291233
http://dx.doi.org/10.1038/s42003-023-04996-8
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