Plasmids carrying Cas9, as previously described [8 (link)], were digested using the XbaI enzyme (New England Biolabs) and incubated for 3 h at 37 °C. The digested plasmids were recovered and purified. In vitro transcription was performed using the T7 high yield RNA Synthesis Kit (New England Biolabs), followed by RNA purification with the GeneJET RNA Purification Kit (Thermo). After purification, the 5’ end of the synthesized Cas9 mRNA was capped using the Vaccinia Capping System (New England Biolabs). The concentration of the capped product was determined after purification and stored at −80 °C for use.
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