Third, we used catalytically dead Cas9 (dCas9) with three different guide RNAs (gRNAs) at the site of the SNP to attempt CRISPR interference, or CRISPRi (Qi et al., 2013) in HEK 293T cells (C/C, major/major at rs2277862). The rationale was that if the variant were truly functional and lay within a transcriptional regulatory element, then the presence of the bulky Cas9 protein at the site should sterically hinder recruitment of transcription factors to the regulatory site and thus interfere with transcriptional regulation. We found that the gRNAs generally resulted in decreased expression of CPNE1 but not ERGIC3 (Figure 3D).
