HiPlex BreakTag strategy. Previously reported genomic Cas9 target sequences (ref. 7 ) were bioinformatically split into 10 pools, each containing approximately 150 sequences. A T7 promoter sequence was added to the 5′ end of each sgRNA protospacer, and a Cas9 sgRNA scaffold sequence was added at the 3′ end by a PCR assembly reaction, which generates a dsDNA template for T7 IVT. T7-transcribed sgRNAs were used for BreakTag with Cas9 in gDNA from HepG2 cells.
