CRISPR-Cas9 Modulation of IL1RN Gene

HEK293T cells were transfected using Lipofectamine 3000 (Invitrogen) with 255 ng gRNA expression plasmid (63.75 ng of plasmid DNA encoding each of four gRNAs targeting IL1RN or the off-target control HBG1) and 245 ng of pcDNA3.1-CRY-dCas9-VP64. Primer sequences and standard curves were previously described (20 (link)). IL1RN gRNAs were CR1 (TGTACTCTCTGAGGTGCTC, at –29), CR2 (ACGCAGATAAGAACCAGTT, at –180), CR3 (CATCAAGTCAGCCATCAGC, at –113), CR4 (GAGTCACCCTCCTGGAAAC). HBG1 control gRNAs were CR1 (GCTAGGGATGAAGAATAAA, –26), CR2 (TTGACCAATAGCCTTGACA, –101), (TGCAAATATCTGTCTGAAA, –163), (AAATTAGCAGTATCCTCTT, –209). As a negative control cells were transfected with 500 ng of an empty pCMV plasmid. Cells were either illuminated with blue light using a custom-built 6 × 4 LED array (1 s pulses every 15 s, 450 nm, 16 mW/cm²) starting 4 h after transfection or incubated in the dark for the entire experiment. Three days after transfection total mRNA was purified using Qiagen RNeasy spin prep columns. cDNA was synthesized using the SuperScript® VILO cDNA Synthesis Kit (Life Technologies). Relative levels of cDNA were detected using Quanta PerfeCta® SYBR^® Green FastMix^® (Quanta Biosciences) and CFX96 Real-Time PCR Detection System (Bio-Rad). Raw data was normalized to GAPDH levels and cells transfected with an empty plasmid control using the ΔΔCT method.