The Effect of Cell Cycle Progression and a Nuclear Localization Signal on CRISPR/Cas9 Gene Correction
Summary
The CRISPR/Cas9 system enables site-specific genome-editing which predominantly occurs via Non-Homologous End Joining (NHEJ), a relatively error-prone pathway. Another pathway is gene correction via Homology Directed Repair (HDR), which can be favoured by presenting an HDR template concomitantly with the Cas9 protein and sgRNA to the cell. Nuclear localization signals (NLSs) can subsequently promote transport of this cargo, encapsulated in lipid nanoparticles (LNPs), into the nucleus. In this work, nuclear localization during different cell cycle stages was determined using a fluorescence-ubiquitin cell cycle indicator (FUCCI) system. Tracking nuclear localization of fluorescently labelled Cas9 over time using confocal microscopy showed that Cas9 enters nuclei predominantly during S/G2/M. Interestingly, the nuclear localization signal did not seem to play a significant role in this process. Even in the presence of ivermectin (0-300μM), a drug inhibiting the process induced by NLS SV40, no significant differences were seen in the occurrence of HDR and NHEJ between samples with and without the NLS. The next goal is to increase the efficiency of nuclear localization of Cas9 through other methods. The human protein phosphatase II (IPP-2) protein for example is primarily taken up during S phase and could thereby stimulate Cas9 uptake during S/G2/M, resulting in an increase in nuclear uptake of Cas9.