Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination
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RESEARCH ARTICLE
Open Access
Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination Sanum Bashir1,2†, Tu Dang1†, Jana Rossius1, Johanna Wolf3 and Ralf Kühn1*
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
Background The RNA guided Cas9 nuclease is a versatile tool for genome editing in mammalian cells by creation of targeted double-strand breaks (DSBs) [1]. Gene editing at Cas9 induced DSBs is achieved by two alternative DSB repair pathways, either by non-homologous end joining (NHEJ) that leads to randomly sized small deletions or insertions (Indels), or by homology-directed repair (HDR) enabling precise sequence modifications that are copied from a repair template molecule. Since HDR is restricted to the S and G2 phases of the cell cycle [2] and requires the presence of a repair template it occurs notably less frequently than NHEJ, presenting a barrier for all applications that rely on precise sequence * Correspondence: [email protected] † Sanum Bashir and Tu Dang contributed equally to this work. 1 Max-Delbrück-Centrum für Molekulare Medizin, 13125 Berlin, Germany Full list of author information is available at the end of the article
modifications, such as modelling of disease mutations or the correction of mutations in somatic gene therapy. To reinforce precise gene editing, tools or interventions are required that bias DSB repair pathway choice in favor of HDR and that promote HDR processing by the targeted delivery of DNA repair templates to DSBs. In particular the availability of repair templates may present a rate limiting factor for HDR. Previous approaches for the targeted delivery of repair templates used Cas9 fusion proteins with domains binding to a functional group that is incorporated into synthetic oligonucleotides or PCR frag
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