C03 FAN1 prevents crispr-CAS9 nickase-induced contractions of CAG/CTG repeats

Abstract

Background Longer CAG repeats in the HTT gene causes more severe forms of Huntington’s disease (HD), suggesting that contracting them would provide a much needed therapeutic benefit. We developed the first method to contract expanded repeats in human cell lines, whilst avoiding concomitant expansions. Unlike other systems that induce double strand breaks around the repeat tract to remove the repeat region, our method relies on the CRIPSR Cas9 nickase. The enzyme is targeted by a guide RNA against the repeat tract, resulting in nicks that are processed by the endogenous DNA repair machinery leading to contractions. The mechanism of contraction is poorly defined and identifying genetic players is important for both improving the contraction efficacy and for stratifying which patients would benefit best from this therapy. Aims Here we tested the hypothesis that loss of FAN1 impacts nickase-induced contractions. Methods/Techniques We generated a HEK293-derived cell line which contains a stably integrated CRISPR-Cas9 nickase, a sgRNA against the repeat tract and an inducible GFP-reporter system containing an ectopic CAG repeat. In this line, we also knocked out FAN1. We assayed for FAN1 loss of function using targeted DNA sequencing, western blotting, and sensitivity to mitomyocin C. We analysed repeat size using flow cytometry for GFP expression and by small-pool PCR. Results/Outcomes We found that two independent FAN1 knockout clones displayed an increase in the frequency of contraction events over 21 days. We found no effect on the frequency of expansion. Conclusions We conclude that FAN1 in our system prevents nickase-induced contractions without impacting expansion events.