In vitro transcription (IVT) of Cas9 mRNA

The open reading frame of SpCas9 was PCR amplified from the pX330 vector and sub-cloned into the pVAX.120 vector consisting of a T7 promoter and 120 bp length of a poly-A tail using standard molecular biology techniques. The IVT reaction was performed in linearized plasmid using T7 RNA polymerase in MEGAscript T7 kit (https://www.thermofisher.com). All mRNAs were produced with an anti-reverse CAP analog (ARCA; [m7G(5′)G]) at the 5′ end (https://www.trilinkbiotech.com/). The IVT-mRNAs were made with following chemical modifications in the indicated ratios: 100% Pseudo-UTP and 25% s2-thio-UTP/5-methyl-CTP (https://www.trilinkbiotech.com/). All IVT mRNAs were purified using the MEGAclear kit (https://www.thermofisher.com) and quantified with nano-photometer and bioanalyzed for quality using the RNA6000 kit in Agilent 2100 Bioanalyzer (https://www.agilent.com).