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A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality
BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining w...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647477/ https://www.ncbi.nlm.nih.gov/pubmed/26578851 http://dx.doi.org/10.1186/s12575-015-0028-4 |
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author | Grassian, A. R. Scales, T. M. E. Knutson, S. K. Kuntz, K. W. McCarthy, N. J. Lowe, C. E. Moore, J. D. Copeland, R. A. Keilhack, H. Smith, J. J. Wickenden, J. A. Ribich, S. |
author_facet | Grassian, A. R. Scales, T. M. E. Knutson, S. K. Kuntz, K. W. McCarthy, N. J. Lowe, C. E. Moore, J. D. Copeland, R. A. Keilhack, H. Smith, J. J. Wickenden, J. A. Ribich, S. |
author_sort | Grassian, A. R. |
collection | PubMed |
description | BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. RESULTS: Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. CONCLUSION: This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12575-015-0028-4) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4647477 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-46474772015-11-18 A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality Grassian, A. R. Scales, T. M. E. Knutson, S. K. Kuntz, K. W. McCarthy, N. J. Lowe, C. E. Moore, J. D. Copeland, R. A. Keilhack, H. Smith, J. J. Wickenden, J. A. Ribich, S. Biol Proced Online Methodology BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. RESULTS: Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. CONCLUSION: This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12575-015-0028-4) contains supplementary material, which is available to authorized users. BioMed Central 2015-11-14 /pmc/articles/PMC4647477/ /pubmed/26578851 http://dx.doi.org/10.1186/s12575-015-0028-4 Text en © Grassian et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 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. |
spellingShingle | Methodology Grassian, A. R. Scales, T. M. E. Knutson, S. K. Kuntz, K. W. McCarthy, N. J. Lowe, C. E. Moore, J. D. Copeland, R. A. Keilhack, H. Smith, J. J. Wickenden, J. A. Ribich, S. A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality |
title | A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality |
title_full | A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality |
title_fullStr | A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality |
title_full_unstemmed | A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality |
title_short | A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality |
title_sort | medium-throughput single cell crispr-cas9 assay to assess gene essentiality |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647477/ https://www.ncbi.nlm.nih.gov/pubmed/26578851 http://dx.doi.org/10.1186/s12575-015-0028-4 |
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