Figure 10.

ABC-KO of target genes by triple-target CRISPR in Cas9 mice. A, Schematic of AAV-3xsgRNA^NeuN that expresses HA-Cre recombinase from hSyn promoter and three U6:sgRNA cistrons. B, Schematic of target site sequences within exons 2 and 4 of NeuN gene. C, Immunoblotting of NeuN proteins in brain lysates from AAV-3xsgRNA^NT, AAV-1xsgRNA^NeuN, AAV-2xsgRNA^NeuN, AAV-3xsgRNA^NeuN (10¹² vg/mice) injected Rosa26^LSL-Cas9 mice. D, Immunoblotting of NeuN proteins in brain lysates from Cas9 mice injected with different doses of AAV-3xsgRNA^NeuN. E, Immunoblotting of NeuN proteins in brain lysates from AAV-3xsgRNA^NeuN injected Cas9 mice at 1, 3, 5, or 7 weeks after (10¹² vg/mice) virus injection. AAV-3xsgRNA^NT injected mouse brains were collected at 3 weeks after virus injection. F, Genomic alignments of whole genome sequencing reads of ABC-NeuN^KO mouse brain DNA at the target sites within exons 2 and 4 of NeuN gene. Top and bottom panels represent read coverage and read alignments, respectively, with different types of mutations highlighted. G, Quantitation of indel mutations at the 3 target sites and 21 predicted off-target sites for the three sgRNAs targeting NeuN gene based on the whole genome sequencing data. H, Immunoblotting of whole-brain lysates from AAV-3xsgRNA^NT and AAV-3xsgRNA^NeuN injected Rosa26^LSL-Cas9 mice with the corresponding antibodies. I, Quantification of the level of NeuN expression in H (n = 4). J, Coimmunostaining of HA-Cre and NeuN in the PFC sections of AAV-3xsgRNA^NT and AAV-3xsgRNA^NeuN injected mice. Bottom row represents magnified images of the corresponding boxed regions in the top row. Scale bars: top, 400 μm; bottom, 100 μm. Data are mean ± SEM. I, Unpaired t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.