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Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System
The nuclease activity of the CRISPR-Cas9 system relies on the delivery of a CRISPR-associated protein 9 (Cas9) and a single guide RNA (sgRNA) against the target gene. CRISPR components are typically delivered to cells as either a Cas9/sgRNA ribonucleoprotein (RNP) complex or a plasmid encoding a Cas...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8978543/ https://www.ncbi.nlm.nih.gov/pubmed/35386234 http://dx.doi.org/10.3389/fgeed.2022.854866 |
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author | Shalaby, Karim E. Aouida, Mustapha Gupta, Vijay Ghanem, Simona S. El-Agnaf, Omar M. A. |
author_facet | Shalaby, Karim E. Aouida, Mustapha Gupta, Vijay Ghanem, Simona S. El-Agnaf, Omar M. A. |
author_sort | Shalaby, Karim E. |
collection | PubMed |
description | The nuclease activity of the CRISPR-Cas9 system relies on the delivery of a CRISPR-associated protein 9 (Cas9) and a single guide RNA (sgRNA) against the target gene. CRISPR components are typically delivered to cells as either a Cas9/sgRNA ribonucleoprotein (RNP) complex or a plasmid encoding a Cas9 protein along with a sequence-specific sgRNA. Multiple transfection reagents are known to deliver CRISPR-Cas9 components, and delivery vectors are being developed for different purposes by several groups. Here, we repurposed a dual-fluorescence (RFP-GFP-GFP) reporter system to quantify the uptake level of the functional CRISPR-Cas9 components into cells and compare the efficiency of CRISPR delivery vectors. Using this system, we developed a novel and rapid cell-based microplate reader assay that makes possible real-time, rapid, and high throughput quantification of CRISPR nuclease activity. Cells stably expressing this dual-fluorescent reporter construct facilitated a direct quantification of the level of the internalized and functional CRISPR-Cas9 molecules into the cells without the need of co-transfecting fluorescently labeled reporter molecules. Additionally, targeting a reporter gene integrated into the genome recapitulates endogenous gene targeting. Thus, this reporter could be used to optimize various transfection conditions of CRISPR components, to evaluate and compare the efficiency of transfection agents, and to enrich cells containing desired CRISPR-induced mutations. |
format | Online Article Text |
id | pubmed-8978543 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-89785432022-04-05 Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System Shalaby, Karim E. Aouida, Mustapha Gupta, Vijay Ghanem, Simona S. El-Agnaf, Omar M. A. Front Genome Ed Genome Editing The nuclease activity of the CRISPR-Cas9 system relies on the delivery of a CRISPR-associated protein 9 (Cas9) and a single guide RNA (sgRNA) against the target gene. CRISPR components are typically delivered to cells as either a Cas9/sgRNA ribonucleoprotein (RNP) complex or a plasmid encoding a Cas9 protein along with a sequence-specific sgRNA. Multiple transfection reagents are known to deliver CRISPR-Cas9 components, and delivery vectors are being developed for different purposes by several groups. Here, we repurposed a dual-fluorescence (RFP-GFP-GFP) reporter system to quantify the uptake level of the functional CRISPR-Cas9 components into cells and compare the efficiency of CRISPR delivery vectors. Using this system, we developed a novel and rapid cell-based microplate reader assay that makes possible real-time, rapid, and high throughput quantification of CRISPR nuclease activity. Cells stably expressing this dual-fluorescent reporter construct facilitated a direct quantification of the level of the internalized and functional CRISPR-Cas9 molecules into the cells without the need of co-transfecting fluorescently labeled reporter molecules. Additionally, targeting a reporter gene integrated into the genome recapitulates endogenous gene targeting. Thus, this reporter could be used to optimize various transfection conditions of CRISPR components, to evaluate and compare the efficiency of transfection agents, and to enrich cells containing desired CRISPR-induced mutations. Frontiers Media S.A. 2022-03-21 /pmc/articles/PMC8978543/ /pubmed/35386234 http://dx.doi.org/10.3389/fgeed.2022.854866 Text en Copyright © 2022 Shalaby, Aouida, Gupta, Ghanem and El-Agnaf. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Genome Editing Shalaby, Karim E. Aouida, Mustapha Gupta, Vijay Ghanem, Simona S. El-Agnaf, Omar M. A. Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System |
title | Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System |
title_full | Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System |
title_fullStr | Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System |
title_full_unstemmed | Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System |
title_short | Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System |
title_sort | rapid assessment of crispr transfection efficiency and enrichment of crispr induced mutations using a dual-fluorescent stable reporter system |
topic | Genome Editing |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8978543/ https://www.ncbi.nlm.nih.gov/pubmed/35386234 http://dx.doi.org/10.3389/fgeed.2022.854866 |
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