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. Author manuscript; available in PMC: 2022 Dec 28.
Published in final edited form as: Nat Genet. 2022 Feb 3;54(2):170–179. doi: 10.1038/s41588-021-00993-x

Extended Data Fig. 3. Knock-in of the rs3824662 risk A allele in GM12878 cell by using CRISPR/Cas9 editing.

Extended Data Fig. 3

a, CRISPR design for knock-in. A 120nt template single-strand DNA containing rs3824662 A allele and flanking sequence was used as the donor for homology-directed repair with CRISPR-Cas9 induced cutting sites. b, Pst1 restriction enzyme is used to screen GM12878 clones with homozygous or heterozygous genotype at rs3824662. c, Sanger sequence results of four successful CRISPR knock-in GM12878 clones. Clones #7 and #49 had knock-in in both alleles; clones #23 and #25 had knock-in in one allele. Experiments were repeated three times independently with consistent results. d, Real time qPCR of GATA3 expression in engineered GM12878 cells with wildtype, heterozygous, or homozygous genotype(n=3) at rs3824662 (p-value = 0.02826 for C/A clones and p-value = 0.001126 for A/A clones by two-sided unpaired t-test). Data are presented as mean values +/− SEM. e, Design and detection of allelic-bias on GATA3 gene expression in GM12878 heterozygous clones. GM12878 cells harbor a nonsynonymous variant (rs2229359 T/C) in GATA3 3rd exon, we performed PCR and Sanger sequencing and observed that the T allele at rs2229359 and A allele at rs3824662 are from the same allele. Therefore, allelic expression derived from rs2229359 would directly inform the differential transcription activation effects of the A vs. C allele at rs3824662 in engineered GM12878 clones. f, Sanger sequencing of PCR products of GATA3 3rd exon cDNA shows allelic expression of GATA3 in two GM12878 heterozygous clone cells by rs2229359 genotyping. g, Shows the transcription level associated with rs3824662-A allele vs. the transcript associated with wild type C allele (n=3) (p value =0.0066 by two-sided unpaired t-test). Data are presented as mean values +/− SEM. h, Western blot of GATA3 and beta-ACTIN in GM12878 wildtype, heterozygous and homozygous clones. Experiments were repeated three times independently with similar results. i, bar plot of relative quantitative value of figure h.