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krCRISPR: an easy and efficient strategy for generating conditional knockout of essential genes in cells
BACKGROUND: CRISPR/Cas9 system is a powerful tool for knocking out genes in cells. However, genes essential for cell survival cannot be directly knocked out. Traditionally, generation of conditional knockout cells requires multiple steps. RESULTS: In this study, we developed an easy and efficient st...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480908/ https://www.ncbi.nlm.nih.gov/pubmed/31049076 http://dx.doi.org/10.1186/s13036-019-0150-y |
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author | Wang, Bei Wang, Zishi Wang, Daqi Zhang, Baolong Ong, Sang-Ging Li, Mingqing Yu, Wenqiang Wang, Yongming |
author_facet | Wang, Bei Wang, Zishi Wang, Daqi Zhang, Baolong Ong, Sang-Ging Li, Mingqing Yu, Wenqiang Wang, Yongming |
author_sort | Wang, Bei |
collection | PubMed |
description | BACKGROUND: CRISPR/Cas9 system is a powerful tool for knocking out genes in cells. However, genes essential for cell survival cannot be directly knocked out. Traditionally, generation of conditional knockout cells requires multiple steps. RESULTS: In this study, we developed an easy and efficient strategy to generate conditional knockout cells by using double episomal vectors – one which expresses gRNA and Cas9 nuclease, and the other expresses an inducible rescue gene. Using this system which we named “krCRISPR” (knockout-rescue CRISPR), we showed that essential genes, HDAC3 and DNMT1, can be efficiently knocked out. When cells reach a desired confluency, the exogenous rescue genes can be silenced by the addition of doxycycline. Furthermore, the krCRISPR system enabled us to study the effects of the essential gene mutations on cells. We showed that the P507L mutation in DNMT1 led to downregulation of global DNA methylation in cells, indicating that it is a disease-causing mutation. CONCLUSIONS: The krCRISPR system offers an easy and efficient platform that facilitates the study of essential genes’ function. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13036-019-0150-y) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6480908 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-64809082019-05-02 krCRISPR: an easy and efficient strategy for generating conditional knockout of essential genes in cells Wang, Bei Wang, Zishi Wang, Daqi Zhang, Baolong Ong, Sang-Ging Li, Mingqing Yu, Wenqiang Wang, Yongming J Biol Eng Research BACKGROUND: CRISPR/Cas9 system is a powerful tool for knocking out genes in cells. However, genes essential for cell survival cannot be directly knocked out. Traditionally, generation of conditional knockout cells requires multiple steps. RESULTS: In this study, we developed an easy and efficient strategy to generate conditional knockout cells by using double episomal vectors – one which expresses gRNA and Cas9 nuclease, and the other expresses an inducible rescue gene. Using this system which we named “krCRISPR” (knockout-rescue CRISPR), we showed that essential genes, HDAC3 and DNMT1, can be efficiently knocked out. When cells reach a desired confluency, the exogenous rescue genes can be silenced by the addition of doxycycline. Furthermore, the krCRISPR system enabled us to study the effects of the essential gene mutations on cells. We showed that the P507L mutation in DNMT1 led to downregulation of global DNA methylation in cells, indicating that it is a disease-causing mutation. CONCLUSIONS: The krCRISPR system offers an easy and efficient platform that facilitates the study of essential genes’ function. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13036-019-0150-y) contains supplementary material, which is available to authorized users. BioMed Central 2019-04-24 /pmc/articles/PMC6480908/ /pubmed/31049076 http://dx.doi.org/10.1186/s13036-019-0150-y Text en © The Author(s). 2019 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 | Research Wang, Bei Wang, Zishi Wang, Daqi Zhang, Baolong Ong, Sang-Ging Li, Mingqing Yu, Wenqiang Wang, Yongming krCRISPR: an easy and efficient strategy for generating conditional knockout of essential genes in cells |
title | krCRISPR: an easy and efficient strategy for generating conditional knockout of essential genes in cells |
title_full | krCRISPR: an easy and efficient strategy for generating conditional knockout of essential genes in cells |
title_fullStr | krCRISPR: an easy and efficient strategy for generating conditional knockout of essential genes in cells |
title_full_unstemmed | krCRISPR: an easy and efficient strategy for generating conditional knockout of essential genes in cells |
title_short | krCRISPR: an easy and efficient strategy for generating conditional knockout of essential genes in cells |
title_sort | krcrispr: an easy and efficient strategy for generating conditional knockout of essential genes in cells |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480908/ https://www.ncbi.nlm.nih.gov/pubmed/31049076 http://dx.doi.org/10.1186/s13036-019-0150-y |
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