Fig. 1: Precise deletion of U12 and U6atac snRNAs by CRISPR-Cas9 system results in defective splicing of U12-type introns and SMA phenotypes in Drosophila.
a Deletion of U12 snRNA and U6atac snRNAs in Drosophila by CRISPR/Cas9 system. Gene loci and deleted regions are indicated. b Validation of deletion strains by northern blotting. Positions and sizes of the U2- and U12-type snRNAs are indicated. c Both the U12Δ/Δ and U6atacΔ/Δ strains are lethal at the pupal stage. Pictures of deletion and WT strains are shown from L3 wandering to pupal stages. d Splicing of U12-type introns are inhibited in deletion strains. Two sets of amplification primers of RT-PCR were used for each intron analysis. Amplification regions are indicated; black lines: U2-type introns; red lines: U12-type introns; boxes: exons. Analyses of other U2- and U12-type introns are shown in Supplementary Fig. 1. e Decreased neuromuscular connection in the U12Δ/Δ and U6atacΔ/Δ strains. Green: neuron visualized by HRP antibody; red: presynaptic and posterior membranes visualized by DLG antibody. Boutons of NMJ in the colocalized regions are counted under microscopy (Smn RNAi: p = 0.00016; U12Δ/Δ: p = 0.00013; U6atacΔ/Δ: p = 9.3e−5). f Hindered larval locomotion of the U12Δ/Δ and U6atacΔ/Δ strains (Smn RNAi: p = 0.00101; U12Δ/Δ: p = 0.00036; U6atacΔ/Δ: p = 0.00019). Path lengths of larval locomotion are recorded and normalized to body lengths. Data represent the mean ± SD from five representatives of each strain, **p < 0.01, ***p < 0.001. p values were calculated using two-sided t-test. Source data are provided as a Source Data file.
