Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks

Abstract

The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks.

Concerning RNA-guided gene drives for the alteration of wild populations

“…Copying efficiencies may also depend on whether the cut produces 5′-overhangs, 3′-overhangs, or blunt ends (Kuhar et al, 2014). Because Cas9 nickases can generate either overhang type while Cas9 nucleases produce blunt ends, the enzyme can be adapted to the needs of the cell type and organism.…”

Near-infrared fluorescent proteins engineered from bacterial phytochromes

“…NIR FPs of the iRFP series are also used in microscopy as additional fluorescent colors, resulting in imaging of multiple FP fusions in a single cell [41, 42]. …”

Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

“…Copying efficiencies may also depend on whether the cut produces 5'-overhangs, 3'-overhangs, or blunt ends 88 . Because Cas9 nickases can generate either overhang type while Cas9 nucleases produce blunt ends, the enzyme can be adapted to the needs of the cell type and organism.…”