Production of Cas9 mRNA and sgRNA

Bicistronic expression vector px330 expressing Cas9, mCherry, and sgRNA was digested with BbsI, and the linearized vector was purified using the Universal DNA Purification Kit (Tiangen). A pair of oligos (Additional file 1: Table S8) for each targeting site was annealed, phosphorylated, and ligated to the linearized vector. The T7 promoter was added to the Cas9 coding region by PCR amplification of px260, using primer Cas9 F and R (Additional file 1: Table S8). The T7-Cas9 PCR product was purified using the Universal DNA Purification Kit (Tiangen) and used as the template for in vitro transcription (IVT) using mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). The T7 promoter was added to the sgRNA template by PCR amplification of px330, using primers listed in Additional file 1: Table S8. The T7-sgRNA PCR product was purified and used as the template for IVT using a MEGA shortscript T7 kit (Life Technologies). Both the Cas9 mRNA and the sgRNAs were purified using a MEGAclear kit (Life Technologies) and eluted in RNase-free water.