Figure 1: Non-homologous DNA increases gene disruption in multiple cell types.

(a) Single- and double-stranded linear non-homologous DNA stimulates indel formation in HEK293T cells. Cas9 was targeted to the EMX1 locus with or without nucleic acid carrier agents (−, no nucleic acid). Indel formation was measured using a T7 endonuclease I assay and is presented as the mean±s.d. of at least two independent experiments (see Supplementary Data 1 for uncropped gels). * denotes significant difference between sample and no nucleic acid (−) control (P<0.05, Welch’s t-test). (b) Non-homologous single-stranded DNA treatment (NOE) increases editing rates in multiple cell types. Editing was performed as described in panel A in multiple cell types either with (dark blue bars, NOE) or without (light blue bars, RNP) 4.5 μg of N-oligo. (c) NOE increases the frequency of homozygous gene disruption. HEK293T cells edited in panel (b) were clonally isolated and amplicons were sequenced to determine genotype. Each horizontal bar represents a single clone with green (wildtype sequence) or magenta (mutations disrupting EMX1) divisions sized according to the percentage of sequencing reads in each category. Zygosity is summarized in the lower table.