a piggyBac transposon containing a puromycin selection cassette, to make PB-rs10889356. For CRISPRi studies, pAC154-dual-dCas9VP160- sgExpression (gift from Dr. Rudolph Jaenisch, Addgene plasmid #48240), a dual expression construct that expresses dCas9-VP160 and sgRNA from separate promoters, was modified by PCR-based methods to remove the VP160 domain and include a viral 2A sequence and GFP after dCas9. Additionally, the gRNA sequence was modified to include a 5-bp hairpin extension, which improves Cas9-gRNA interaction, and a single-basepair substitution (A-U flip) that removes a putative Pol III terminator sequence, as described previously (Chen et al., 2013).
