Abstract
BackgroundPrecise 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.ResultsFor 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.ConclusionsOur findings provide a novel efficient approach to promote precise gene editing in human cells.
Highlights
Precise genetic modifications are preferred products of Clustered regularly interspaced short palindromic repeat (CRISPR)-CRIS PR associated protein 9 (Cas9) mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR)
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
DSB repair assays in traffic light reporter cells To quantitatively determine CRISPR/Cas9-induced DSB repair by HDR or NHEJ, we integrated a ‘traffic light’ reporter (TLR) construct into the Adeno-Associated Virus Integration Site 1 (AAVS1) locus of human HEK293 cells and human induced pluripotent stem cells
Summary
Precise genetic modifications are preferred products of CRISPR-Cas mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Bashir et al BMC Biotechnology (2020) 20:57 of HDR include the enrichment of cells in the S/G2 phase [6, 7], restriction of Cas activity to the S/G2 phase [8, 9], inhibition of NHEJ key molecules [10, 11] and the use of Cas fusion proteins with the HDR effector CtIP [12] These interventions do not directly target the protein complexes determining the repair pathway choice at the DSB ends, that presumably represent an effective target to promote HDR. In contrast to 53BP1, Rad and RNF169 recognize the Ubiquitin mark at H2A-K13 but exhibit substantially lower affinity as compared to the H2AK15Ub site [25]
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