Targeted Transcriptional Regulation using CRISPR-dCas9 Fusions

The constructs, pcDNA-dCas9^{p300 Core} (61357, Addgene)^{38 (link)} pcDNA-dCas9^{p300 Core (D1399Y)} (61358, Addgene)^{38 (link)}, pdCas9^DNMT3A (71666, Addgene)^{39 (link)}, and pdCas9^{DNMT3A (ANV)} (71685, Addgene)^{39 (link)} were from Addgene. gRNAs targeting the MYB promoter and enhancer regions were designed using Feng Zhang lab’s Target Finder software (http://crispr.mit.edu). Best guides, with highest score of the inversed likelihood of off-target binding, were selected, and the gRNA sequences are shown in supplementary Table 1. Expression plasmids for gRNAs were constructed by cloning annealed oligos into pSPgRNA (#47108 Addgene)^{54 (link)} using BbsI (R0539, NEB) and T4 ligase (M0202, NEB). Then these plasmids were transfected into K562 cells using Lipofectamine 3000 (L3000015, Invitrogen) according to the manufacturer’s instructions. Forty-eight hours later, cells were harvested for analysis.