In vitro synthesis of PE2 (nCas9-MMLV RT fusion) polyA mRNA using T7 RNA polymerase
Donald Rio
Abstract
We describe the preparation of synthetic capped and polyadenylated messenger RNA (mRNA) encoding a Cas9 nickase-reverse transcriptase fusion protein (PE2) for use in genome editing with a method called prime editing.
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Steps
Protocol Overview
Preparation of linearized plasmid DNA encoding the PE2 protein for use as a template for in vitro transcription by T7 RNA polymerase.
Synthesis of mRNA by T7 RNA polymerase, using a 5’ cap nucleotide to initiate the mRNA and using E. coli poly A polymerase to add a synthetic poly A tail after transcription.
Column purification of the final synthetic, capped and polyadenylated mRNA.
Part 1: Linearization of pCMV-PE2 plasmid
Digest 100µg plasmid with 200 units Pme I restriction endonuclease in a 1mL reaction using CutSmart buffer for4h 0m 0s. at 37°C.
Purify the cleaved DNA by phenol-chloroform extraction, collect the upper (aqueous) phase and transfer to fresh 1.5mL Eppendorf tubes.
Add 5Molarity (M) NaCl to a final concentration of 0.1Molarity (M) (20µL) and 3 volumes of 100% ethanol.
Leave at-80°C for 2h 0m 0s to 2h 0m 0s to precipitate.
Spin 16000x g,4°C.
Remove the ethanol and air dry for0h 30m 0s.
Resuspend at 500 in TE (10millimolar (mM) Tris-HCl, 7.5, 1millimolar (mM)EDTA) buffer. Store the DNA sample at-20°C.
Part 2: In vitro transcription and polyA tailing
In order get a high yield of the 6500nt long PE2 mRNA by T7 RNA polymerase transcription, set up 8 x 20µL in vitro transcription reactions using 1µg of cleaved template DNA in each reaction and the New England Biolabs HiScribe T7 ARCA kit with tailing (E2060S), according to the manufacturer’s instructions.
Incubate at 37°C for 2h 0m 0s in an incubator (not a temp block).
For each 20µL reaction, add 2µLDNase I (stock is2U/uL).
Incubate at 37°C for 0h 15m 0s.
Dilute the reaction to 50µL with 20µL RNase-free water, 5µL 10X polyA polymerase buffer and 5µL E. coli polyA polymerase.
Incubate at 37°C for 0h 30m 0s.
Part 3: Purification of IVT product
Purify the polyadenylated mRNA on 50µg New England Biolabs Monarch RNA cleanup columns (2 of 20µL reactions per column) (T2040L).
Dilute each 20µL reaction with 100µL binding buffer and 150µL 100% ethanol.
Add the two diluted reactions to one column and spin at 16000x g.
Wash each column with wash buffer and spin twice (see below).
Wash with 500µL wash buffer (spin at 16000x g). (1/2)
Wash with 500µL wash buffer (spin at 16000x g). (2/2)
Elute in25µL of RNase-free H2O per column, incubate for 0h 1m 0s at 37Room temperature.
Spin at 16000x g into fresh 1.5mLtubes and pool elution together into one tube.
Measure the RNA concentration using a Nanodrop1000c spectrophotometer.
Store the mRNA at -80°C and avoid repeated freezing and thawing.
