Cloner supplement

To generate single-cell clones from iPSC derived from GM04723 (i.e., iPSC-C), cells were transfected by electroporation with a lentiCRISPRv2 vector expressing only SpCas9 or a plasmid expressing both SpCas9 and gRNA. Seventy-two hours after puromycin selection (0.5 mg/L), 1.5 × 10⁵ cells were seeded in a 60 mm dish with 10 μM ROCK inhibitor (MilliporeSigma) and then incubated for 2 weeks without ROCK inhibitor. To generate single-cell clones from iPSCs with adult-onset CAG repeats (iPSC-A and iPSC-B), cells were transfected with PX551 EFS and PX552 EFS vectors (containing either EV or the target gRNA) using Lipofectamine Stem Transfection Reagent. Seventy-two hours after transfection without selection, 1.5 × 10⁵ cells were seeded in a 60 mm dish with CloneR supplement (Stemcell Technologies) and then incubated for an additional 2 weeks. Visible colonies were picked and individually maintained in 96-well plates for clonal expansion. Once cells reached approximately 80% confluent, cells were subcultured in two 96-well plates, one for maintenance and the other for genomic DNA extraction to validate the on-target modification by Sanger sequencing analysis. Confirmed targeted clones were then expanded for molecular characterization and RNA-Seq analysis.

Karyotyping was performed on the iPSC line before the CRISPR approach (which we refer to as “parental line” 2), and the results showed a duplication in chromosome 20. The guide RNA 1 and the ribonucleoprotein approach were used to generate APP-KO. A total of 1 × 10⁶ iPSCs were nucleofected with the ribonucleoprotein complex from Integrated DNA Technologies (225 pmol of each RNA and 120 pmol of Cas9 protein). iPSCs were plated 48 hours later at very low density (10 cells/cm²) on Laminin-521 with CloneR supplement (Stem Cell Technology) for clonal selection. One week later, iPSC clones were picked under a stereomicroscope and cultured on Laminin-521 in 96 well plates (Duscher). The resulting iPSC clones were duplicated after confluency and used for cryoconservation and DNA extraction. One base-pair deletion was observed in screening results and the absence of expression of APP protein was confirmed by Western blot. The APP knock-out clone and control were used for further experiments, and the low level of APP mRNA was confirmed by quantitative PCR (fig. S1, L to N). Karyotyping was also performed for the APP-KO clone, control, and parental iPSC. The results confirmed the same duplication in chromosome 22 in all the conditions. No other deletion or duplication was observed in APP-KO clones and control after CRISPR approach.