Complete Disruption of Autism-Susceptibility Genes by Gene Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons

Abstract

Autism spectrum disorder (ASD) is phenotypically and genetically heterogeneous. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of ten ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NGN2)-directed induction of iPSCs allowed production of excitatory neurons, and mutant proteins were not detectable. RNA sequencing revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory postsynaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2-, and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.

Keywords: CRISPR; NGN2; StopTag; autism; convergence; iPSC; isogenic; knockout; sEPSC.

Figure 1

Outline of the Experimental Procedure to Test the Phenotypical Consequences of Gene Repression in iPSC-Derived Glutamatergic Neurons (A) Unaffected human iPSC controls (ctrl) 19-2, labeled in green, were subjected to CRISPR gene editing to introduce a premature termination codon (StopTag), into a target exon of 14 ASD target genes. Knockout (KO) iPSC populations were identified by absolute quantification of StopTag versus wild-type (wt) alleles using droplet digital PCR (ddPCR). Well A1 is an example of a cell population containing 100% wt allele (FAM signal in blue) for a given target locus, while well A2 contains 100% StopTag alleles (VIC signal in green); FAM- and VIC-associated probe sequences are presented in Table S1. KO iPSCs were differentiated into glutamatergic neurons, labeled in red, by means of NGN2 transient overexpression (O/E). Neuronal phenotypes were monitored using RNA-seq, patch-clamp, and multi-electrode array recordings; F and R are ddPCR primers. (B) Full-length integral StopTag sequence insertion was confirmed for all target genes except CHD8, in which the first 39 bp in 5′ of the StopTag sequence were deleted, and ASTN2, in which different point mutations were found (red bars); chr, chromosome; bp, base pair. See also Figures S1 and S2 and Tables S1–S3.

Figure 2

Characterization of the Control 19-2 iPSCs and Neurons (A–D) Representative microscopic images show normal iPSC (A) pluripotency (SSEA4 and OCT4), (B) differentiation potential into the three germ layers in vitro (embryoid bodies: TUBB3, SMA, and AFP) or (C) in vivo (teratoma assays), and (D) karyotype. See also Figure S4. Scale bars: 100 μm (A), 25 μm (B), and 100 μm (C). (E) Representative traces of action potentials recorded at different current injection on the left panel, and the number of action potentials is plotted for each step of current injection on the right panel. (F) Representative traces of sodium currents on the left panel, and currents were recorded at different potentials in voltage-clamp on the right panel; 33 control 19-2 neurons were recorded from three independent differentiation experiments at day 21–28 post-NGN2-induction (PNI). (G) Transcript levels in RPKM of a series of iPSC, neural progenitor cell (NPC), and neuron markers in control 19-2 iPSCs (blue) and control 19-2 glutamatergic neurons (orange). Values are presented as means ± SD of eight independent experiments for iPSCs and four for neurons. See also Figure S3.

Figure 3

Complete KO of Target Gene Expression in Neurons (A) Example of a target locus, i.e., exon 2 of SCN2A, where Spliced Transcripts Alignment to a Reference (STAR) software was used to align reads previously unmapped by TopHat. The gray part of the reads mapped to the human reference genome hg19. The colored part did not map to hg19 but aligned perfectly with the StopTag sequence, showing it is properly transcribed and fused to the target transcripts. (B) Transcript levels in reads per kilobase per million mapped reads (RPKM; y axis) of each target gene (x axis) in their corresponding KO iPSCs (black bars) and KO neurons (red bars), as well as in control iPSCs (gray bars) and control neurons (blue bars). Values are presented as means ± SD of 2–8 independent experiments; ^∗FDR <0.05 in neurons. See also Table S4. (C) Western blots showing the absence of the major form of ATRX (upper panel) and SCN2A (bottom panel) proteins in their corresponding KO neurons, compared with control 19-2 neurons, 4 weeks PNI; ACTB, loading control. (D) Western blots revealing the absence of any truncated form of proteins in different KO neuron lines, using a V5 antibody. Predicted (kDa), predicted size of potentially truncated peptides based on the insertion sites of the StopTag within each target transcript relative to the start codon position; n/a, not applicable; ACTB and TUBB3, loading controls; n/a, not available.

Figure 4

Transcriptional Characterization of KO iPSCs and Neurons (A) Enrichment map of differentially expressed genes in different mutant iPSCs compared with the isogenic control 19-2, as revealed by RNA-seq and pathway analysis. For example, the upper right piece of each pie represents the SCN2A-null iPSC line (see inset), in which different gene sets associated with “embryonic morphogenesis” are upregulated (red color), with respect to the control 19-2 line. The color intensity correlates with the Benjamini-Hochberg false discovery rate (BH-FDR) values, as depicted in the inset. Values were calculated from (n) independent experiments; n = 8 for ctrl 19-2; n = 4 for ATRX^−/y, KCNQ2^−/−, SCN2A^−/−, and ASTN2^−/−; n = 2 for AFF2^−/y. (B) Differentially expressed genes in different KO neurons compared with the isogenic control 19-2 as revealed by RNA-seq. Values are presented as log2 fold change. Values were calculated from (n) independent differentiation experiments; n = 5 for AFF2^−/y; n = 4 for ctrl 19-2, ATRX^−/y, KCNQ2^−/−, SCN2A^−/−; n = 3 for ASTN2^−/−. (C) RNA-seq validation of transcript levels of seven genes in three different AFF2-null neuron lines, i.e., 19-2-AFF2^−/y-1, 19-2-AFF2^−/y-2, and 50B-AFF2^−/y, compared with their respective control lines 19-2 and 50B. Values are presented as means ± SD of (n) independent differentiation experiments where n = 4 for controls 19-2 and 50B; n = 5 for 19-2-AFF2^−/y-1; n = 3 for 19-2-AFF2^−/y-2 and 50B-AFF2^−/y; ^∗each KO line value has an FDR <0.5 compared with its respective control line. See also Figure S4 and Tables S5–S7.

Figure 5

Electrophysiological Phenotyping of KO iPSC-Derived Neurons (A) Representative traces of excitatory postsynaptic current (EPSC; top panel); spontaneous EPSC frequency (lower left panel) and amplitude (lower right panel) were recorded from different KO neurons; total number of recorded neurons is indicated on the graphs; values are presented as means ± SEM of three independent differentiation experiments for all, except two for AFF2 and KCNQ2, recorded at day 21–28 PNI. (B) Representative raster plots over a 5-min recording of multi-electrode array experiments (top panels); mean firing rate (lower left panel), burst frequency (lower middle panel), and network burst frequency (lower right panel) were recorded for the five significant genes from the sEPSC frequency graph in (A). Each spike is represented with a short black line. A burst was considered as a group of at least five spikes, each separated by an inter-spike interval (ISI) of no more than 100 ms. A network burst (pink lines) was identified as a minimum of ten spikes with a maximum ISI of 100 ms, occurring on at least 25% of electrodes per well. From 21 to 55 different wells were recorded per line, with usually 6–8 wells per line per experiment. Values are presented as means ± SEM from (n) independent differentiation experiments, where n = 8 for ctrl 19-2; n = 6 for ATRX^−/y; n = 5 for SCN2A^−/−; n = 4 for AFF2^−/y, ASTN2^−/−, and KCNQ2^−/−; recorded at week 8 PNI; ^∗p < 0.05 from one-way ANOVA (Dunnett multiple comparison test). n/d, not detected. See also Figure S5.