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| Item Type: | Article |
|---|---|
| Title: | Enhancement of CRISPR-Cas9 induced precise gene editing by targeting Histone H2A-K15 ubiquitination |
| Creators Name: | Bashir, S., Dang, T., Rossius, J., Wolf, J. and Kuehn, R. |
| Abstract: | 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. |
| Keywords: | Precise Gene Editing, CRISPR, Cas9, Genome Editing, Rad18, RNF169, BRCA1, tetR, Gal4, HR, Animals |
| Source: | BMC Biotechnology |
| ISSN: | 1472-6750 |
| Publisher: | BioMed Central |
| Volume: | 20 |
| Number: | 1 |
| Page Range: | 57 |
| Date: | 23 October 2020 |
| Official Publication: | https://doi.org/10.1186/s12896-020-00650-x |
| PubMed: | View item in PubMed |
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