Extended Data Fig. 3: Detection of insertion-deletion mutations introduced by the CRISPR-Cas9 system, and Nts mRNA depletion.
From: Neurotensin orchestrates valence assignment in the amygdala
a–d: To compute the percentage of insertion-deletion (indel) mutations introduced into the Nts gene by the CRISPR-Cas9 system in vivo, we harvested tissue from viral-injection sites (AAV-Cas9 + AAV-Nts guide 1 or AAV-Cas9 + AAV-Nts guide 2 in the MGN), sorted the nuclei using fluorescence, extracted the genomic DNA, used PCR to amplify the parts of the DNA targeted by each guide and used next-generation sequencing (NGS) to sequence the DNA. For the control group, tissue was harvested from the same brain region, but from either the hemisphere contralateral to the viral injection, or the same brain region in an un-injected mouse. The percentage of indel mutations were quantified using the website outknocker.org (a, c). For the Guide 1 and Guide 2 groups in (a) and (c) respectively, only one tissue sample was derived from each injected mouse. For the control groups, the 4 and 5 samples in (a) and (c) were derived from 2 and 3 mice, respectively. There were significantly higher number of indel mutations in the Cas9+Guide 1 (a; unpaired t-test, two-tailed, t8 = 85.99, ***P < 0.0001, effect size = 90.82 ± 1.056, CI95 = 88.38 to 93.25) and Cas9+Guide 2 (c; unpaired t-test, two-tailed, t7 = 21.20, ***P < 0.0001, effect size = 80.27 ± 3.787, CI95 = 71.32 to 89.23) injected tissue, compared with mutations in the control tissue. (b,d)The percentage of reads from a representative mouse in each of the Guide 1-injected (b) and Guide 2-injected (d) groups are shown. N denotes number of tissue samples in each group. Error bars around the mean indicate s.e.m. e–f: Quantification of Nts mRNA and Vglut2 mRNA using FISH, where both guides were co-introduced into the brain. Data shown here are from separate sets of mice that received an AAV-Cas9 injection along with an injection of either AAV-Nts guide 1 (e) or AAV-Nts guide 2 (f). Using Cas9 in combination with guide 1 depleted Nts mRNA levels (e, left; paired t-test, t5 = 3.251, *P = 0.0227, effect size = 0.1950, CI95 = 0.04078 to 0.3492), while Vglut2 mRNA levels were not affected (e, right; paired t-test, t5 = 0.5491, P = 0.6065, effect size = 0.3050, CI95 = −1.123 to 1.733). Using Cas9 in combination with guide 2 depleted Nts mRNA levels (f, left; paired t-test, t5 = 3.193, *P = 0.0242, effect size = 0.4850, CI95 = 0.09443 to 0.8756), while Vglut2 mRNA levels were not affected (f, right; paired t-test, t5 = 1.280, P = 0.2568, effect size = 0.5617, CI95 = −0.5666 to 1.690). N denotes number of mice in each group. Error bars around the mean indicate s.e.m.
