Editing Cleancap mRNA Invitro Transcription (section)

== Cas13a ==

In vitro transcription of Cas13a. Plasmids were linearized with Not-I HF (New England Biolabs) overnight at 37 °C. Linearized templates were purified by sodium acetate (Thermo Fisher Scientific) precipitation and rehydrated with nuclease-free water. In vitro transcription was performed overnight at 37 °C using the HiScribe T7 Kit (NEB) following the manufacturer’s instructions (N1-methyl-pseudouridine modified). The resulting RNA was treated with DNase I (Aldevron) for 30 min to remove the template, and it was purified using lithium chloride precipitation (Thermo Fisher Scientific). The RNA was heat denatured at 65 °C for 10 min before capping with a Cap-1 structure using guanylyl transferase and 2′-O-methyltransferase (Aldevron). mRNA was then purified by lithium chloride precipitation, treated with alkaline phosphatase (NEB) and purified again. mRNA concentration was measured using a Nanodrop. mRNA stock concentrations were 1–3 mg ml−1. Purified mRNA products were analyzed by gel electrophoresis to ensure purity. crRNA guides were purchased from Integrated DNA Technologies or GenScript [https://www.aldevron.com/hubfs/content/Documents/ald-IVT-enzyme-tech-guide-090010916.pdf https://www.aldevron.com/hubfs/content/Documents/ald-IVT-enzyme-tech-guide-090010916.pdf]