Extended Data Fig. 4: In vivo validation of CRISPR-Cas9 mediated Nts gene cKO.
From: Neurotensin orchestrates valence assignment in the amygdala
a: The CRISPR-associated endonuclease Cas9 was used to generate indels in the Nts gene for depleting NT levels in specific NT populations. NT-specific single guide RNAs were designed to target exon 1 and 3 of the NT coding region. b: Experimental design to validate the efficacy of the CRISPR-Cas9 system in depleting Nts mRNA levels. Four weeks after steretotaxic injection of AAV9 viral vectors encoding Cas9 and both the guides into the right MGN, brains were extracted and mRNA levels of Nts and Vglut2 transcripts were assessed using FISH. c: Confocal images from a representative brain slice showing fluorescence from the nuclear stain DAPI, GFP in the Nts guides, FISH of Nts mRNA and Vglut2 mRNA from the control (top) and Cas9+guide injected hemispheres (bottom). d: Representative images showing overlap between the GFP-KASH and Cas9 mRNA expressing neurons. We used a multi-AAV viral system to deliver the different components of the CRISPR-Cas9 gene editing system (Cas9 and guides) to neurons in vivo. Since gene editing requires the Cas9 protein and one or more guides to be present in the same cell, we quantified the amount of overlap between cells expressing the Cas9 and the guide components. The images shown here are from a representative mouse. The bottom row is a magnified version of the area indicated by the solid white square in the top row. e: Quantification of penetrance of the AAV vectors carrying Cas9 and the guides. Both the Cas9 protein and guide RNAs targeting the Nts gene need to be present in a cell for the Cas9 protein to edit the Nts gene. f: Percentage of fluorescent pixels was used as a metric to quantify Nts and Vglut2 mRNA levels. Nts mRNA levels were significantly reduced in the Cas9+guide injected hemisphere, compared to the non-injected hemisphere (paired t-test, t5 = 3.315, *P = 0.0211, effect size = 0.4983, CI95 = 0.1118 to 0.8849; left panel), while there was no detectable difference in the levels of Vglut2 mRNA between the two hemispheres (paired t-test, t5 = 1.442, P = 0.2089, effect size = 0.4283, CI95 = −0.3355 to 1.192; right panel). N denotes number of mice in each group. Error bars around the mean indicate s.e.m.
