HEK293t CRISPRi

HEK293t cells (ATCC) were maintained in DMEM (Gibco # 11995-065) supplemented with 10% FBS and 1% penicillin–streptomycin (10,000 U/ml; Gibco #15140122) solution at 37 °C in 5% CO₂. Four guide RNAs were designed for each targeted region by CHOPCHOP or MIT’s guide design tool based on maximizing cutting efficiency, minimizing off-targets, and closest proximity to the region of interest. Guide sequences are provided in the Supplementary Data. Guides were then individually cloned using golden gate methodology into the guide vector upstream of an eGFP gene. Cells were transfected into HEK293t cells at 50–70% confluency using Lipofectamine LTX with the 4 guide vectors and dCas9 vector (dCas9–KRAB upstream of BFP fluorophore) at a ratio of three parts Cas9 to one part guide, where each individual guide was added in equal amounts to additional guides for that region. After 48 hours, double-positive GFP and BFP fluorescing cells were collected into culture media via FACs sorting, spun down, and frozen. Sorting was performed on a BD FACS Aria Fusion 5–18 using BD FACSDiva 8.0.1 software. In the case of the Cas9 only control population, BFP single positive cells were sorted out of the population via FACS and frozen. This was repeated 4–5 times to have technical replicates. RNA was extracted using the Qiagen RNeasy RNA mini extraction kit and 500 ng of RNA was used as input for RNA-seq. RNA-seq was performed using the NEB Next Ultra II Directional RNA-seq kit. Data analysis: Reads were mapped and gene counts were quantified with STAR (v2.5.1a). Counts were filtered to retain autosomal genes and exclude lowly expressed genes (<1 CPM). PC1 clearly associated with the sorting batch, so the sorting batch was added as a covariate into the final linear model. p Values were identified using glmQLFTest() from edgeR. Because of the very small effect sizes of CRISPRi in non-coding regions, cis-genes within the SBK1-ATP2A1 loci were considered for final significance testing. This list included all protein-coding genes within these loci passing the expression threshold as well as GAPDH (14 genes total). Raw p values from these genes were Bonferroni corrected to get adjusted p values (p < 0.05/14 genes). Only genes passing the Bonferroni significance threshold were considered significantly affected by the CRISPRi perturbations.