Scarless genome editing of induced pluripotent stem cells (iPSCs) is crucial for the precise modeling of genetic disease. Here we present CRISPR Del/Rei, a two-step deletion-reinsertion strategy with high editing efficiency and simple PCR-based screening that generates isogenic clones in ~ 2 months. We apply our strategy to edit iPSCs at 3 loci with only rare off target editing.

The polygenic nature of schizophrenia (SCZ) implicates many variants in disease development. Rare variants of high penetrance have been shown to contribute to the disease prevalence. Whole-exome sequencing of a large three-generation family with SCZ and bipolar disorder identified a single segregating novel, rare, non-synonymous variant in the gene CASKIN1. The variant D1204N is absent from all databases, and CASKIN1 has a gnomAD missense score Z = 1.79 and pLI = 1, indicating its strong intolerance to variation. We find that introducing variants in the proline-rich region where the D1204N resides results in significant cellular changes in iPSC-derived neurons, consistent with CASKIN1’s known functions. We observe significant transcriptomic changes in 368 genes (padj < 0.05) involved in neuronal differentiation and nervous system development. We also observed nominally significant changes in the frequency of action potentials during differentiation, where the speed at which the edited and unedited cells reach the same level of activity differs. Our results suggest that CASKIN1 is an excellent gene candidate for psychosis development with high penetrance in this family.

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DPYSL2/CRMP2 is a microtubule-stabilizing protein crucial for neurogenesis and associated with numerous psychiatric and neurodegenerative disorders. DPYSL2 has multiple RNA and protein isoforms, but few studies have differentiated between them or explored their individual functions. We previously demonstrated in HEK293 cells that a schizophrenia-associated variant in the DPYSL2 B isoform (DPYSL2-B) reduced the length of cellular projections, created a transcriptomic disturbance that captured schizophrenia etiology, and was acted upon by the mTOR pathway. In the present study, we follow up on these results by creating, to our knowledge, the first models of endogenous DPYSL2-B knockout in human induced pluripotent stem cells and excitatory glutamatergic neurons. We use CRISPR/Cas9 to specifically knock out DPYSL2-B and observe corresponding reduction of its RNA and protein. The average length of dendrites in knockout neurons was reduced up to 58% compared to controls. Transcriptome analysis reveals disruptions in pathways highly relevant to psychiatric disease including mTOR signaling, cytoskeletal dynamics, immune function, calcium signaling, and cholesterol biosynthesis. We also observed a significant enrichment of our differentially expressed genes in schizophrenia GWAS-associated loci. Our findings clarify the functions of the human DPYSL2-B isoform and confirm its involvement in molecular pathologies shared between many psychiatric diseases.