Journal: Cell stem cell
Article Title: Large diverse population cohorts of hiPSCs and derived hepatocyte-like cells reveal functional genetic variation at blood lipid-associated loci
Figure Lengend Snippet: Evidence for rs2277862- CPNE1 as a Functional SNP-Gene Set (A) Schematics of human chromosome 20q11 locus showing the relative positions of rs2277862, CPNE1 , and ERGIC3 and mouse chromosome 2qH1 locus showing the relative positions of rs27324996, Cpne1 , and Ergic3 . (B) Top panels: heterozygous knock-in of rs2277862 minor allele with a single-strand DNA oligonucleotide. Representative indels in non-knock-in clones are also shown. Bottom panels: homozygous 38-bp deletions (“knockout” or Δ38/Δ38) encompassing rs2277862 using a dual gRNA approach. A representative agarose gel of PCR amplicons is shown. The protospacers are underlined, the PAMs are bolded, and the SNP position is indicated in red. (C) Left panels: gene expression in undifferentiated HUES 8 cells (n = 2 wild-type clones and 1 knock-in clone; 6 wells per clone) and differentiated HUES 8 HLCs (n = 2 wild-type clones and 1 knock-in clone; 6 wells per clone) normalized to mean expression level in wild-type clones. Right panels: gene expression in undifferentiated HUES 8 cells (n = 10 wild-type and 10 knockout clones, 3 wells per clone) and undifferentiated H7 cells (n = 8 wild-type and 6 knockout clones, 3 wells per clone) normalized to mean expression level in wild-type clones. (D) CRISPR interference at the rs2277862 site. The three gRNA protospacers are underlined, the PAMs are bolded, and the SNP position is indicated in red. The graphs show gene expression, normalized to mean expression level in control cells, in HEK 293T cells transfected with catalytically dead Cas9 (dCas9) with the gRNAs (either singly or in combination, 3 wells per condition). Control cells were transfected with dCas9 without an accompanying gRNA. (E) The noncoding rs2277862 site is well conserved in mouse, including allelic variants of the SNP itself, with the murine equivalent being rs27324996. The SNP position is indicated in red, non-conserved nucleotides are indicated in blue, the gRNA protospacer used to generate the knock-in mouse is underlined, and the PAM is bolded. The electropherogram is from a mouse in which the minor allele of rs2277862/rs27324996 (T) has been knocked into one chromosome, along with four non-conserved nucleotides to “humanize” the site. (F) Gene expression in liver from littermates of the C57BL/6J background (n = 18 wild-type mice and 10 homozygous knock-in mice), normalized to mean expression level in wild-type mice. Data are displayed as means and s.e.m. P -values were calculated with two-tailed Welch’s t -tests.
Article Snippet: The region flanking rs27324996 was PCR amplified (F: 5′-TGGGAATGGCTTCTTAGGGC-3′ and R: 5′-CATCCCCAAGCAACTCAACC-3′) using AccuPrime Taq DNA Polymerase (Thermo Fisher Scientific) with the following cycling conditions: 94°C for 2 min, [94°C for 30 sec, 55°C for 30 sec, 68°C for 30 sec] × 40 cycles, 68°C for 5 min. PCR products were purified using the DNA Clean & Concentrator kit (Zymo Research) and analyzed for the presence of indels using the Surveyor Mutation Detection Kit (Integrated DNA Technologies) according to the manufacturer’s instructions.
Techniques: Functional Assay, Knock-In, Clone Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Expressing, Knock-Out, CRISPR, Transfection, Mouse Assay, Two Tailed Test