Despite advances in gRNA design algorithms (Chari et al., 2015, Doench et al., 2014, Doench et al., 2016, Xu et al., 2015), gRNA efficacy across individuals, cell types, and dCas9 effectors still must be empirically confirmed. We report clear cell line-dependent effects in gRNA efficacy and hypothesize that epigenetic differences drive this variability across our experiments. There is substantial evidence that the epigenetic landscape impacts gRNA efficacies, including chromatin structure (Chari et al., 2015, Knight et al., 2015, Singh et al., 2015, Wu et al., 2014), nucleosome positioning (Horlbeck et al., 2016b), and DNA methylation (Wu et al., 2014). Histone modifications or competitive interactions with transcriptional machinery may also affect target site accessibility, although so far this has not been as well investigated. Overall, it seems that Cas9 activity is greatest at sites of open chromatin; because chromatin remodeling can restore Cas9 access (Horlbeck et al., 2016b), it is possible that chromatin-modifying enzymes may improve efficacy, although the impact of such treatment on disease-modeling experiments is unknown. It is also possible that alternative and/or combinations of dCas9 effectors acting further
