Characterizing triple point-mutations and generation of Syt1^M3 and Syt2^M3 KI mice.

A. Sequence alignment of the BoNT-binding region in Syt1 and Syt2 (residues 32 to 52 in Syt1 and 40 to 60 in Syt2). Residues highlighted in red mark the designed triple mutations (Syt1^M3 and Syt2^M3) that abolish BoNT-binding. B. Co-crystal structure of the BoNT/B-Syt2 complex (PDB:4KBB) with the triple mutation sites (F54A, F55A, E57K) highlighted. C-E. Miniature inhibitory postsynaptic currents (mIPSC) were monitored and analyzed by whole-cell patch-clamp recording in cultured rat cortical neurons, with representative traces shown in C, frequency (freq) in D, and amplitude (amp) in E. WT: wild type neurons; Syt1 KD: neurons with their endogenous Syt1 knocked down by shRNA expressed via lentiviral transduction; Syt1^WT: WT Syt1 is expressed in Syt1 KD neurons via lentiviral transduction; Syt1^M3: Syt1^M3 mutant was expressed in Syt1 KD neurons via lentiviral transduction. Syt1 KD increased mIPSC frequency, which is restored by expression of either Syt1^WT or Syt1^M3. For each condition, we recorded from 12–15 neurons from a total of four coverslips. Data shown are means ± SEM. Statistical analysis was performed with Student’s t-test (***P < 0.001). F-H. Spontaneous inhibitory postsynaptic currents (sIPSC) were monitored and analyzed by whole-cell patch-clamp recording in cultured rat cortical neurons, with representative traces shown in F, frequency in G, and amplitude in H. The amplitude and frequency of sIPSC were greatly deceased in Syt1 KD, and both Syt1^WT and Syt1^M3 restored normal levels of sIPSC. Data shown are means ± SEM. Statistical analysis was performed with Student’s t-test (***P < 0.001). I-K. Evoked inhibitory postsynaptic currents (IPSC) were monitored and analyzed by whole-cell patch-clamp recording in cultured rat cortical neurons, with representative traces shown in I, amplitude in J, and total charge transfer in K. The amplitude and charge of IPSC were greatly deceased in Syt1 KD, and both Syt1^WT and Syt1^M3 restored normal levels of IPSC. L. Schematic drawing of generating Syt1 and Syt2 triple mutation KI mice via CRISPR-Cas9 approach. M. Expression levels of synaptic vesicle proteins Syt1, Syt2, and SV2, and toxin substrate proteins SNAP-25, VAMP-2, Syntaxin-1, analyzed by immunoblot of brain lysates, remain unchanged in Syt1^M3 KI and Syt2^M3 KI mice compared with WT mice.