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. 2023 Jul 13;12:RP86972. doi: 10.7554/eLife.86972

Figure 9. Physiological requirement of SLC6A17 for glutamine (Gln) transport into synaptic vesicles (SVs) in vivo.

(A) A schematic diagram illustrating the strategy of Cas9-mediated cleavage of Slc6a17 specifically in Slc6a17-positive neurons, and simultaneous labeling of all SVs in these neurons by Syp-HA. (B) Immunoblot results showing SLC6A17 protein was significantly reduced in targeted neurons, while Syp-HA was efficiently tagged onto the SVs of these neurons. (C) Volcano plot of contents of SVs from AAV-sgRNA/Slc6a17iCre/Cas9+ targeted neurons compared to contents of SVs from control (AAV-sgRNA /Slc6a17iCre/Cas9-) neurons. Glu, GABA, ACh, and the nine previously reported substrates of SLC6A17 are listed. (D) Ratios of the level of a molecule from the SVs of AAV-sgRNA /Slc6a17iCre/Cas9+ neurons vs. the level of the same molecule from SVs of AAV-sgRNA/Slc6a17iCre/Cas9- neurons shown as fold change (log2 transformed). GABA level was significantly increased (p=0.0034 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-). Gln level was significantly decreased (p<0.0001 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-). (E–H) Contents of SVs from Slc6a17 containing neurons were quantified to mole per 10 μl HA beads (n = 18, 27 for Slc6a17iCre/Cas9- and Slc6a17iCre/Cas9+, respectively, from six and nine different animals with three replicates each): Glu (E, p=0.0005 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-); GABA (F, p=0.0032 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-); Gln (G, p<0.0001 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-); Ser (H, p=0.0979 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-).

Figure 9—source data 1. Data points for Figure 9C-I.
Figure 9—source data 2. Original files of the full raw unedited blots for Figure 9B.
Figure 9—source data 3. Uncropped blots with the relevant bands labeled for Figure 9B.

Figure 9.

Figure 9—figure supplement 1. CRISPR/Cas9-mediated Slc6a17 gene cleavage in Slc6a17 expressing cells of adult mice.

Figure 9—figure supplement 1.

(A) A schematic diagram illustrating the strategy for Cas9-mediated cleavage of Slc6a17 specifically in Slc6a17-positive neurons, and simultaneous labeling of SVs by Syp-HA. A single tRNA-gRNA allowed multiple gRNAs being efficiently produced, which could be precisely excised in vivo by the endogenous RNases, to improve editing efficiency of Cas9 system. (B) SLC6A17 protein in Slc6a17iCre/Cas9+ mice were efficiently removed. The protein level of SLC6A17 was measured in the input supernatant used for immunoisolation.
Figure 9—figure supplement 1—source data 1. Original files of the full raw unedited blots for Figure 9—figure supplement 1B.
Figure 9—figure supplement 1—source data 2. Uncropped blots with the relevant bands labeled for Figure 9—figure supplement 1B.