All CRISPR gRNAs used in SGE and essentiality experiments were cloned into pX45924 (link). This plasmid expresses the gRNA from a U6 promoter, as well as a Cas9–2A-puromycin resistance (-puroR) cassette. S. pyogenes Cas9 target sites were chosen for SGE experiments on multiple criteria, assessed in the following order: 1.) To induce cleavage within BRCA1 coding sequence, 2.) To target a genomic site permissive to synonymous substitution within the guanine dinucleotide of the PAM or the protospacer, 3.) To have minimal predicted off-target activity47 (link), 4.) To have maximal predicted on-target activity48 (link).
Complementary oligos ordered from Integrated DNA Technologies (IDT) were annealed, phosphorylated, diluted and ligated into BbsI-digested and gel-purified pX459, as described24 (link). Ligation reactions were transformed into E. coli (Stellar competent cells, Takara), which were plated on ampicillin. Colonies were cultured and Sanger sequenced to confirm correct gRNA sequences. Purification of sequence-verified plasmids for transfection was performed with the ZymoPure Maxiprep kit (ZymoResearch). For targeting LIG4 in HAP1 cells, pX45824 (link) was used instead of pX459, which expresses EGFP in lieu of puroR.
Complementary oligos ordered from Integrated DNA Technologies (IDT) were annealed, phosphorylated, diluted and ligated into BbsI-digested and gel-purified pX459, as described24 (link). Ligation reactions were transformed into E. coli (Stellar competent cells, Takara), which were plated on ampicillin. Colonies were cultured and Sanger sequenced to confirm correct gRNA sequences. Purification of sequence-verified plasmids for transfection was performed with the ZymoPure Maxiprep kit (ZymoResearch). For targeting LIG4 in HAP1 cells, pX45824 (link) was used instead of pX459, which expresses EGFP in lieu of puroR.
