Fourth, we noted that, atypically, the noncoding region encompassing rs2277862 is well conserved in mouse (Figure 3A and 3E), and the orthologous nucleotide in mouse also displays naturally occurring variation and has been previously cataloged as rs27324996, with the same two alleles (C and T) as in humans. We used CRISPR-Cas9 in single-cell mouse embryos of the C57BL/6J strain (C/C at rs27324996) to generate a knock-in mouse with a minor allele (T) as well as four additional nucleotide changes both to prevent CRISPR-Cas9 re-cleavage of the knock-in allele and to “humanize” the sequence (i.e., make a perfect match to the orthologous human sequence) (Figure 3E). There was decreased expression of Cpne1 (down 37%) and Ergic3 (down 34%, albeit not with P < 0.05) in the liver in homozygous knock-in (T/T) mice compared to wild-type (C/C) littermates (Figure 3F); cholesterol levels were unchanged, as expected from the small effect in humans (1.19 mg/dL change in cholesterol per allele). These data are highly concordant with the data from the genome-edited hPSC clones with respect to both the direction and magnitude of effects. Taken together, all of these findings support rs2277862-CPNE1 as a functional SNP-gene set.
