Tyr loss of function enables the selection of crispants with the highest mutation rate while keeping other behavioral and developmental parameters unchanged. (A): Dorsal (lower panels) and lateral (upper panels) images of scrambled (left panels) and tyrosinase crispants (right panels), with a reduction in pigmentation in the latest. (B–D): Analysis and comparison of different relevant morphological phenotypes in both crispants (scrambled are represented in purple; tyrosinase crispants are represented in blue. The error bar represents the minimum to maximum values): body length (µm) (B), the diameter of the eyes (µm) (C) and heart area (µm²) (D). (E–G): Analysis and comparison of the most relevant parameters related to epilepsy in both crispants (scrambled are represented in purple; tyrosinase crispants are represented in blue. Error bar represents the minimum to maximum values): distance moved (mm) during the dark/light cycles phase (E), maximum velocity achieved after the light flashes (mm/s) (F) and the number of angle turns after the light flashes (G). (H): Bar plot showing the mutagenesis efficiency observed in the targeted loci in pigmented larvae (dark blue) and unpigmented larvae (light blue). From left to right: mutation rate observed in the adrgr1 CDS in adrgr1 crispants; mutation rate observed in the gabra1 CDS in gabra1 crispants; mutation rate observed in the kcnq2a CDS in kcnq2a single crispants; mutation rate observed in the kcnq2a CDS in kcnq2a-kcnq2b double crispants; mutation rate observed in the kcnq2b CDS in kcnq2b single crispants; mutation rate observed in the kcnq2b CDS in kcnq2a-kcnq2b double crispants; mutation rate observed in the pcdh19 CDS in pcdh19 crispants; mutation rate observed in the scn1lab CDS in scn1lab crispants; mutation rate observed in the ube3a CDS in ube3a crispants. Error bars represent the standard error of the mean (SEM), **** p < 0.001 (t-test).

Characterization of adgrg1 and gabra1 crispants. (A–D). Representation of adgrg1 measurements in behavioral studies: distance moved (mm) during the dark/light cycles (A), the maximum velocity (mm/s) during the light phase in response to different PTZ concentrations (DMSO, PTZ 1 mM and PTZ 3 mM) (B), representative tracking images of scrambled (left) and adgrg1 crispants (right) after the light stimuli to induce convulsions (C) and the maximum speed (mm/s) and angle turns measures after the light stimuli (D). (E–H). Representation of gabra1 measurements in behavioral studies: distance moved (mm) during the dark/light cycles (E), the maximum velocity (mm/s) during the light phase in response to different PTZ concentrations (DMSO, PTZ 1 mM and PTZ 3 mM) (F), representative tracking images of scrambled (left) and gabra1 crispants (right) after the light stimuli to induce convulsions (G) and the maximum speed (mm/s) and angle turns measures after the light stimuli (H). Scrambled are represented in blue, adgrg1 crispants are represented in dark blue and gabra1 crispants are represented in orange. The error bar represents the minimum to maximum values. ** p < 0.01, **** p < 0.001 (t-test). Next, we decided to test the possibility of inducing epileptic-like behavior by exposing the crispants to different epileptogenic stimuli. In order to trigger seizures, we employed two kinds of stimuli, namely incubation with Pentylenetetrazole (PTZ) and exposure to intermittent flashes of light. PTZ is a pro-convulsant commonly used as a pharmacological epilepsy model [25,28,39,40,41] while flashing or flickering lights are known to cause photosensitive seizures in a large number of patients affected by epilepsy [20].

Characterization of kcnq2a, kcnq2b, double kcnq2a/kcnq2b and pcdh19 crispants. (A–D). Representation of kcnq2a, kcnq2b and double kcnq2a/kcnq2b measurements in behavioral studies: distance moved (mm) during the dark/light cycles (A), the maximum velocity (mm/s) during the light phase in response to different PTZ concentrations (DMSO, PTZ 1 mM and PTZ 3 mM) (B), representative tracking images of scrambled (top left), kcnq2a crispants (top right), kcnq2b crispants (bottom left) and double kcnq2a/kcnq2b crispants (bottom right) after the light stimuli to induce convulsions (C), and the maximum speed (mm/s) and angle turns measures after the light stimuli (D). (E–H). Representation of pcdh19 measurements in behavioral studies: distance moved (mm) during the dark/light cycles (E), the maximum velocity (mm/s) during the light phase in response to different PTZ concentrations (DMSO, PTZ 1 mM and PTZ 3 mM) (F), representative tracking images of scrambled (left) and pcdh19 crispants (right) after the light stimuli to induce convulsions (G) and the maximum speed (mm/s) and angle turns measures after the light stimuli (H). Scrambled are represented in blue, kcnq2a crispants are represented in red, kcnq2b crispants are represented in pink, double kcnq2a/kcnq2b crispants are represented in light pink and pcdh19 crispants are represented in orange. The error bar represents the minimum to maximum values. * p < 0.05, *** p < 0.005, **** p < 0.001 (t-test).

Characterization of scn1lab and ube3a crispants. (A–D). Representation of scn1lab measurements in behavioral studies: distance moved (mm) during the dark/light cycles (A), the maximum velocity (mm/s) during the light phase in response to different PTZ concentrations (DMSO, PTZ 1 mM and PTZ 3 mM) (B), representative tracking images of scrambled (left) and scn1lab crispants (right) after the light stimuli to induce convulsions (C) and the maximum speed (mm/s) and angle turns measures after the light stimuli (D). (E–H). Representation of ube3a measurements in behavioral studies: distance moved (mm) during the dark/light cycles (E), the maximum velocity (mm/s) during the light phase in response to different PTZ concentrations (DMSO, PTZ 1 mM and PTZ 3 mM) (F), representative tracking images of scrambled (left) and ube3a crispants (right) after the light stimuli to induce convulsions (G) and the maximum speed (mm/s) and angle turns measures after the light stimuli (H). Scrambled are represented in blue, scn1lab crispants are represented in light blue and ube3a crispants are represented in green. The error bar represents the minimum to maximum values. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001 (t-test).

The identification of crispants with the greatest photosensitivity can be obtained through a multiparametric analysis of their reaction to light flashes. (A): Bidimensional plot of the different variables selected as more relevant to perform the principal component analysis (PCA), with a vectorial representation. The selected variables are maximum velocity (mm/s), the number of angle turns, maximum acceleration (mm/s²), angular velocity (deg/s), mobility in the arena (%) and the cumulative duration (s) of three different mobility states, immobile, mobile and highly mobile. (B): PCA biplot comparing two main groups, the scrambled (in black) and the different studied crispants related to childhood epileptic genes (in blue). (C): The definition of two different populations depending on their activity through the calculation of Mahalanobis distance. The low activity group (in green) represents the population of analyzed larvae with a non-epileptic behavior; the high activity group (in orange) represents the population of analyzed larvae with an epileptic-like behavior in response to light flashes. (D): PCA biplot comparing the two main groups described through the Mahalanobis distance analysis, considering the different PCA variables. (E): A representation of the percentage of larvae classified in the epileptic-like population of the scrambled and all the selected crispants.

Incubating scn1lab and gabra1 crispants with antiepileptic compounds offers protection against seizure-like events. (A): Tracking plots of the different crispants 2 s after the light flashes. Scale bar = 6 mm. The different plots correspond (from left to right) to scrambled, scn1lab crispants treated with DMSO (vehicle), scn1lab crispants treated with two different concentrations of fenfluramine (17.5 µM and 35 µM), topiramate (50 µM and 100 µM) and valproic acid (50 µM and 100 µM). (B–D): Representation of the percentage of larvae classified in the high-activity region. Scrambled treated with DMSO are represented in dark blue in all plots, scn1lab crispants treated with DMSO are represented in light blue in all plots. (B): scn1lab crispants treated with fenfluramine 17.5 µM are represented in light purple; scn1lab crispants treated with fenfluramine 25 µM are represented in dark purple. (C). scn1lab crispants treated with topiramate 50 µM are represented in light pink; scn1lab crispants treated with topiramate 100 µM are represented in dark pink. scn1lab crispants treated with valproic acid 50 µM are represented in light green; scn1lab crispants treated with valproic acid 100 µM are represented in dark green. In all bar graphs, * p < 0.05 (binomial test).