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Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination
BACKGROUND: Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathw...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7585302/ https://www.ncbi.nlm.nih.gov/pubmed/33097066 http://dx.doi.org/10.1186/s12896-020-00650-x |
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author | Bashir, Sanum Dang, Tu Rossius, Jana Wolf, Johanna Kühn, Ralf |
author_facet | Bashir, Sanum Dang, Tu Rossius, Jana Wolf, Johanna Kühn, Ralf |
author_sort | Bashir, Sanum |
collection | PubMed |
description | BACKGROUND: Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology. RESULTS: For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci. CONCLUSIONS: Our findings provide a novel efficient approach to promote precise gene editing in human cells. SUPPLEMENTARY INFORMATION: Supplementary information accompanies this paper at 10.1186/s12896-020-00650-x. |
format | Online Article Text |
id | pubmed-7585302 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-75853022020-10-26 Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination Bashir, Sanum Dang, Tu Rossius, Jana Wolf, Johanna Kühn, Ralf BMC Biotechnol Research Article BACKGROUND: Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology. RESULTS: For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci. CONCLUSIONS: Our findings provide a novel efficient approach to promote precise gene editing in human cells. SUPPLEMENTARY INFORMATION: Supplementary information accompanies this paper at 10.1186/s12896-020-00650-x. BioMed Central 2020-10-23 /pmc/articles/PMC7585302/ /pubmed/33097066 http://dx.doi.org/10.1186/s12896-020-00650-x Text en © The Author(s) 2020 Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. |
spellingShingle | Research Article Bashir, Sanum Dang, Tu Rossius, Jana Wolf, Johanna Kühn, Ralf Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination |
title | Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination |
title_full | Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination |
title_fullStr | Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination |
title_full_unstemmed | Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination |
title_short | Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination |
title_sort | enhancement of crispr-cas9 induced precise gene editing by targeting histone h2a-k15 ubiquitination |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7585302/ https://www.ncbi.nlm.nih.gov/pubmed/33097066 http://dx.doi.org/10.1186/s12896-020-00650-x |
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