Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes

Abstract

We demonstrate editing of post-mitotic neurons in the adult mouse brain following injection of Cas9 ribonucleoprotein (RNP) complexes in the hippocampus, striatum and cortex. Engineered variants of Cas9 with multiple SV40 nuclear localization sequences enabled a tenfold increase in the efficiency of neuronal editing in vivo. These advances indicate the potential of genome editing in the brain to correct or inactivate the underlying genetic causes of neurological diseases.

Figure 1.. Generation of cell penetrating Cas9 RNPs.

A) Images and flow cytometry analysis of tdTomato⁺ NPC neurospheres 3 days post-nucleofection of Cas9 RNP dose course. Scale bar 400μm. Representative data from n=3 independent experiments. B) N-terminal 1–7xNLS-Cas9–2xNLS design; C) Direct delivery of 1–7xNLS-Cas9–2xNLS with NPCs led to activation of tdTomato reporter in genome-edited cells. 4xNLS-Cas9–2xNLS designs are more efficient at genome-editing cells than other designs. Data are represented as Tukey box and whisker plots, box is IQR, whiskers are the lowest datum still within 1.5 IQR of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile, outliers are open circles, + is mean, line in median(100pmol 0xNLS vs. 4xNLS; Two-tailed Unpaired t test with Welch’s correction; p = 0.0236 and F_3,3= 80.50. 100pmol 0xNLS vs. 4xNLS-noGFP; Two-tailed Unpaired t test with Welch’s correction; p = 0.0015 and F_5,3 = 39.04. 100pmol 1xNLS vs. 4xNLS-noGFP; Two-tailed Unpaired t test with Welch’s correction; p = 0.0099 and F_5,3 = 2.434. 100pmol 0xNLS vs. 7xNLS; Two-tailed Unpaired t test with Welch’s correction; p = 0.0283 and F_3,3= 5.246. 10pmol 0xNLS vs. 2xNLS; Two-tailed Unpaired t test with equal SD; p = 0.0096 and F_2,2 = 2.597. 10pmol 0xNLS vs. 4xNLS-noGFP; Two-tailed Unpaired t test with equal SD; p = 0.0462 and F_3,2 = 4.072) n=4 experimental replicates with 2 technical replicates each. D) Genomic DNA PCR of tdTomato STOP locus validates tdTomato⁺ flow cytometry analysis. 4xNLS-Cas9–2xNLS RNP complexes yield deletion edits (lower 2 bands) while 0xNLS-Cas9–2xNLS RNP complexes do not. 100pmol 4xNLS-Cas9–2xNLS RNP bottom DNA band (activated tdTomato locus) contains 5.4% +/− 0.4% of the total DNA correlating with %tdTomato⁺ cells observed in 1C. Representative gel from n=4 experimental replicates with 2 technical replicates each.

Figure 2.. Injection of Cas9 RNP into multiple brain regions in adult mice

A) Color-coded dots indicate stereotaxic injection sites on coronal cartoons of mouse brain. Single Cas9 RNP injections (4pmol/0.5μl) with (Cas9 construct; sgRNA) 0xNLS-Cas9–2xNLS;sgRNA-tdTom, 4xNLS-Cas9–2xNLS;sgRNA-non-targeting, 4xNLS-Cas9–2xNLS;sgRNA-tdTom or sham (injection buffer only) into hippocampus, striatum, primary somatosensory cortex (S1), primary visual cortex V1. Male mice are 14–15 weeks old. B) Quantification of tdTomato⁺ cells / pmol RNP delivered. Brains were analyzed 12–14 days post injection. 50μm thick floating sections, 1 section every 300μm analyzed. 4xNLS-Cas9–2xNLS RNPs are significantly more efficient compared to 0xNLS-Cas9–2xNLS RNPs for in vivo genome-editing in all brain regions tested. Sham and 4xNLS-Cas9–2xNLS;non-targeting RNPs do not activate tdTomato indicating specificity of genome editing with Cas9 RNPs. Data are presented as mean ± SEM (4xNLS;tdTom vs. 0xNLS;tdTom; Two-tailed Unpaired t test with equal SD; p = 0.002 and F_5,5 = 5.9 for the striatum. Two-tailed Unpaired t test with Welch’s correction p = 0.03 and F_{5, 5} = 215 for Cortex S1. p = 0.03 and F_{5, 5} = 25 for Cortex V1. p = 0.04 and F_{5, 5} = 83 for the hippocampus.) The sample size for each group is as follows: Sham and 4xNLS-Cas9–2xNLS;non-targeting: n=2 animals, n=2 injections per animal for each group, 0xNLS-Cas9–2xNLS;tdTom: n=3 animals, n=2 injections per animal, 4xNLS-Cas9–2xNLS;tdTom: n=3 animals, n=2 injections per animal. C) Representative confocal microscopy images of 4xNLS-Cas9–2xNLS RNP treated cells identify tdTomato⁺ cells co-localizing with neuron and not astrocyte marker proteins. NeuN, neuronal specific nuclear protein in vertebrates. CTIP2, aka BCL11a, a transcription factor present in CA1 hippocampus and striatum neurons. DARPP-32, cAMP-regulated neuronal phosphoprotein, a marker of striatum medium spiny neurons. GFAP, glial fibrillary acidic protein, an intermediate filament protein expressed in astrocytes and ependymal cells of the CNS. S100β, a highly expressed protein in striatal astrocytes. Scale bar is 50μm. D) Increasing dose of 4xNLS-Cas9–2xNLS RNP significantly increases number of tdTomato⁺ genome-edited cells in the striatum. Red dots indicate bilateral stereotaxic injection sites on coronal cartoon of mouse brain. Coronal section mosaic tile image of bilateral 30pmol RNP injections with tdTomato reporting genome-editing in the striatum. Blue=DAPI staining nuclei, Red=endogenous tdTomato expression. E) Representative confocal images of tdTomato⁺ cells in single 4, 15, 30pmol/0.5μl injection dose course. Scale bar 100μm. F) Quantification of total # tdTomato⁺ cells per injection site. Data are presented as Tukey box and whisker plots. Box is IQR, whiskers are the lowest datum still within 1.5 IQR of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile, + is mean, line is median Two-tailed Unpaired t test with Welch’s correction p = 0.0188 and F_{5, 5} = 46.03 for 4pmol v.30pmol. p = 0.0536 and F_{5, 5} = 13.43 for 4pmol v. 15pmol. Two-tailed Unpaired t test with equal SD; p = 0.0845 and F_{5, 5} = 3.428 for 15pmol v.30pmol. n=3 animals, n=2 injections per animal for each group. G) Quantification of tdTomato⁺ cells per pmol RNP delivered (1 tdTomato⁺ cell per 10fmol RNP). Data are presented as Tukey box and whisker plots. Box is IQR, whiskers are the lowest datum still within 1.5 IQR of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile, + is mean, line is median (p = 0.213; Kruskal-Wallis test. n=3 animals, n=2 injections per animal for each group).