Extended Data Fig. 5: Confirmation of KO efficiency of TERF2 and LNPK and effects of TERF2 loss on interneuron saltatory migration.
From: Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment
a, Estimation of gene perturbation effect sizes. Log2 fold change enrichments, normalized to the median of negative ‘safe’-targeting controls, of sgRNAs between the Dlxi1/2::eGFP+ and Dlxi1/2::eGFP− samples are plotted in the indicated color; the distribution of safe-targeting sgRNA controls is shown in black. The dashed line is the casTLE-estimated maximum effect size. b–c, Genotype analysis of hiPS cells (b) or day 40 hSO (c) showing the PCR amplicon of individual candidate genes. These experiments have been repeated 3 times. d–e, NGS analysis of amplicons amplified from genomic DNA extracted from day 40 hSO derived from TERF2 KO (d) and LNPK KO (e) cells. The percentage of each amplicon that is more than 2% is presented. In (d), TERF2 116’ has an additional 21 bp insertion. f–i, Representative western blotting images and quantifications showing TERF2 (f, g) and LNPK (h, i) abundance in KO and Cas9-CTL hSO (day 60–80, n = 3 samples). GAPDH was used as a loading control. j, Percentage of Dlxi1/2b::GFP+ cells in day–57 hSO (n = 8 from 2 differentiations). Each organoid was processed as one sample. Kruskal-Wallis test (P = 0.1316). k, hSO size over time (from 4 differentiations). mixed model two-way ANOVA (genotype as factor, F2,42 = 3.329) using Dunnett’s multiple comparison test. * P = 0.0324. l, Left, a representative immunofluorescence image showing LNPK expression in hSO. Right, inset showing LNPK co-localizes with SEC61B-mEGFP in hSO derived from SEC61B-mEGFP hiPS cells. This experiment has been repeated 3 times. m, Saltatory migration of Dlxi1/2b::eGFP+ cells in the hCO side of the Cas9-CTL and TERF2 KO hFA. Triangles mark the nucleus and arrows mark the dilation that moves before nuclear translocation. n–p, Saltation length, number of saltations, and speed of movement of Dlxi1/2b::eGFP+ cells in the hCO side of the hFA (Cas9-CTL, n = 47 cells from 14 hFA; TERF2 KO, n = 26 cells from 8 hFA, from 3-4 differentiations). n, **** P < 0.0001, two-tailed unpaired t-test. o, p, Two-tailed Mann-Whitney test. o, P = 0.5497, p, * P = 0.0267. Data are presented as mean ± s.e.m. (g, i, j, k, n–p).
