The rs3749171 mutation in the human GPR35 gene was generated by a single-base substitution (C > T) using CRISPR-Cas9–induced homology-directed repair in the KOLF2 human iPSC line. This was achieved by nucleofection of 106 cells with Cas9-crRNA-tracrRNA ribonucleoprotein complexes. Synthetic RNA oligonucleotides (target site: 5′-CCTGGTCACGGCCATCGCCG-3′ or 5′-CACATAGCGGTCCACGGCGA-3′, 225 pmol of crRNA/tracrRNA) were annealed by heating to 95°C for 2 min in duplex buffer [Integrated DNA Technologies (IDT)] and cooling slowly, followed by addition of 122 pmol of recombinant eSpCas9_1.1 protein [in 10 mM tris-HCl (pH 7.4), 300 mM NaCl, 0.1 mM EDTA, and 1 mM dithiothreitol], incubation at room temperature for 20 min, and addition of 500 pmol of a 100-nucleotide single-stranded DNA oligonucleotide (IDT Ultramer) as a homology-directed repair template to introduce the desired base change. After recovery, plating at single-cell density, and colony picking into 96-well plates, 480 clones were screened for heterozygous and homozygous mutations by high-throughput sequencing of amplicons spanning the target site using an Illumina MiSeq instrument. Final cell lines were further validated by Sanger sequencing. Two independently targeted clones homozygous for 108T or 108M were used in downstream assays.

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