Supplementary Figure 3: Cas9 RNP variants are equally active in mitotic and post-mitotic cells when assisted across the cell membrane but only 4xNLS-Cas9-2xNLS is active in post-mitotic cortical neurons under direct delivery conditions.

A) Cas9 RNP complexes targeting d2EGFP are mixed with Lipofectamine2000 and delivered to HEK293T-d2EGFP cells. Percent EGFP gene disruption is measured by FACS analysis. RNPs mixed with Lipofectamine2000 resulted in robust GFP disruption and 0xNLS-Cas9-2xNLS was not significantly different from 4xNLS-Cas9-2xNLS at all RNP doses. Data are represented as mean ± SD (4xNLS-Cas9;sgRNA-NT3 vs. 0xNLS-Cas9;sgRNA-NT3; Two-tailed Unpaired t test with equal SD; p = 0.9032 and F_{5, 5} = 1.034). n=3 experimental replicates with 3 technical replicates each. B) NaCl hypertonic protein transduction protocol to deliver RNPs into tdTomato NPCs²³. Editing activity for 4xNLS-Cas9-2xNLS-sfGFP, 0xNLS-Cas9-2xNLS-sfGFP, 0xNLS-Cas9-2xNLS and Cas9 with no NLS was not significantly different at all RNP doses. (2way ANOVA with Tukey’s multiple comparisons test; p=0.8685 and F_9,19=0.4825). n=3 experimental replicates with 2 technical replicates each. C) We assessed the ability of 4xNLS-Cas9-2xNLS and 0xNLS-Cas9-2xNLS to edit primary cortical neurons (which are post-mitotic and no longer breakdown the nuclear envelope during cell division) in cell culture by direct delivery of RNP complexes or by LNP carrier assisted delivery, RNP+Lipofectamine2000. The efficiency of neuron editing was low in all cases, <0.05% of all seeded cells were tdTomato⁺ / MAP2⁺ or NeuN⁺ (marker proteins of post-mitotic mature neurons). Direct delivery of 4xNLS-Cas9-2xNLS results in edited post-mitotic neurons (NeuN⁺/tdTomato⁺) while 0x-NLS-Cas9-2xNLS does not. n=3 experimental replicates with 2 technical replicates each. D) In contrast, when RNPs were mixed with Lipofectamine2000 both 4xNLS-Cas9-2xNLS and 0xNLS-Cas9-2xNLS RNPs triggered genome editing in mature neurons (MAP2⁺, tdTomato⁺). Therefore, both 4xNLS-Cas9-2xNLS and 0xNLS-Cas9-2xNLS RNPs are capable of editing post-mitotic neurons if assisted across the cell membrane by Lipofectamine2000, but only 4xNLS-Cas9-2xNLS is capable of penetrating and triggering genome editing in neurons when directly added to the media. n=3 experimental replicates with 2 technical replicates each. Scale bar 10μm.