CRISPR-Cas9 sgRNA Design and Synthesis

sgRNAs were designed using the CHOP-CHOP online utility (http://chopchop.cbu.uib.no/). sgRNA targeting sites are shown in Fig. 1. As described in a recent publication [23 (link)], the DNA template for T7 promoter used to drive in vitro transcription was constructed by PCR. Briefly, a customized oligonucleotide containing the T7 promoter and the sgRNA target sequence (N₂₀ or N₁₈) was designed as a forward primer with the sequence 5′-TAATACGACTCACTATAGG(N₂₀ or N₁₈)GTTTTAGAGCTAGAAATAGC. The T7 promoter sequences are underlined. The reverse primer was 5′-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3′. sgRNA synthesis was performed using a RiboMax large scale RNA production system—T7 kit (Promega, cat. P1300), following the manufacturer’s instructions.