Extended Data Figure 3.

A. Selected GO terms for biological processes for SYNGAP1 immunoprecipitation data collected from D.I.V. 7 cortical organoids.

B. Schematic of line generation details for isogenic control of Patient^p.Q503X.

C. Chromatogram of the generated corrected line (Patient^Corrected). The truncating “T” was substituted with the wild type “C” base pair.

D. Representative Western blot for SYNGAP1 in the Patient ^p.Q503X(P), Patient^Corrected (C) and KO (K) iPSCs derived neurons showing a reduction of SYNGAP1 levels in P and complete loss in K iPSCs

E. Quantification of the western blot shows significant reduction in SYNGAP1 levels in Patient ^p.Q503X iPSCs compared to the Patient^Corrected iPSCs in four biological replicates. Individual dots represent independent replicates. Data are presented as mean values± SD. Statistical analysis was performed using unpaired two-tailed t-test. P value <0.01.

F. Quantification of the SYNGAP1 peptide. Graphical representation of SYNGAP1 protein levels quantified by timed parallel reaction monitoring (tRPM). Plot shows a decrease of SYNGAP1 total protein levels in Patient^p.Q503X:64.83 (51.6%) as compared to its corresponding isogenic control (Patient^Corrected): 125.6 expressed as fg peptide/ug digested protein. Individual dots represent 20 organoids pooled together. N=9 across three independent differentiations. Data are presented as mean values ± SD. Statistical analysis was performed using unpaired two-tailed t-test. P value <0.0001.

G. Karyotypic analysis of Patient^Corrected iPSCs revealed a normal karyotype.

H. Chromatogram of the Patient^p.Q503X iPSCs carrying the truncating mutation.

I. PCR of RGD, Patient^p.Q503X, 03231 cell line.

J. Chromatogram of the 03231^Control iPSCs carrying the wild type sequence

K. Chromatogram of the 03231^RGD iPSCs carrying the homozygous mutation in the RGD domain.

L. Karyotypic analysis of 03231^RGD iPSCs revealed a normal karyotype.

M. Chromatogram of the 03231^p.Q503X iPSCs carrying the truncating mutation.

N. PCR of 03231^Control cell line and 03231^p.Q503X cell line showing haploinsuffiency in the 03231^p.Q503X line.

O. Karyotypic analysis of 03231^p.Q503X iPSCs revealed a normal karyotype.