Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9

Abstract

CRISPR/Cas9 is the most commonly used genome editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR/Cas9 system to control exposure of the Cas9 protein NLS. Although blue light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR/Cas9 editing system without the NLS and a CRISPR/Cas9 editing system containing an NES were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the hitchhiking of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene editing systems, as well as provide new insights into the evolution of the nucleus and of compatible protein functions.