TITLE: In vitro synthesis of PE2 (nCas9-MMLV RT fusion) polyA mRNA using T7 RNA polymerase
AUTHORS: Donald  Rio

[DESCRIPTION]
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.

[STEPS]
SECTION: Protocol Overview
1. Preparation of linearized plasmid DNA encoding the PE2 protein for use as a template for in vitro transcription by T7 RNA polymerase.
SECTION: Protocol Overview
2. 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.
SECTION: Protocol Overview
3. Column purification of the final synthetic, capped and polyadenylated mRNA. 
SECTION: Part 1: Linearization of pCMV-PE2 plasmid
4. Digest 100 µg plasmid with 200 units Pme I restriction endonuclease in a 1 mL reaction using CutSmart buffer for240 min. at 37 °C.

SECTION: Part 1: Linearization of pCMV-PE2 plasmid
5. Purify the cleaved DNA by phenol-chloroform extraction, collect the upper (aqueous) phase and transfer to fresh 1.5 mL Eppendorf tubes.

SECTION: Part 1: Linearization of pCMV-PE2 plasmid
6. Add 5 Molarity (M) NaCl to a final concentration of 0.1 Molarity (M) (20 µL) and 3 volumes of 100% ethanol.

SECTION: Part 1: Linearization of pCMV-PE2 plasmid
7. Leave at-80 °C for  120 min to 120 min to precipitate.

SECTION: Part 1: Linearization of pCMV-PE2 plasmid
8. Spin 16000 x g, 10 min, 4 °C.

SECTION: Part 1: Linearization of pCMV-PE2 plasmid
9. Remove the ethanol and air dry for30 min.

SECTION: Part 1: Linearization of pCMV-PE2 plasmid
10. Resuspend at 500 1809 in TE (10 millimolar (mM) Tris-HCl, pH 7.5, 1 millimolar (mM)EDTA) buffer.  Store the DNA sample at-20 °C.

SECTION: Part 2: In vitro transcription and polyA tailing
11. In order get a high yield of the 6500nt long PE2 mRNA by T7 RNA polymerase transcription, set up 8 x 20 µL in vitro transcription reactions using 1 µg 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.

SECTION: Part 2: In vitro transcription and polyA tailing
12. Incubate at 37 °C for 120 min in an incubator (not a temp block).

SECTION: Part 2: In vitro transcription and polyA tailing
13. For each 20 µL reaction, add 2 µLDNase I (stock is2 U/uL).

SECTION: Part 2: In vitro transcription and polyA tailing
14. Incubate at 37 °C for 15 min.

SECTION: Part 2: In vitro transcription and polyA tailing
15. Dilute the reaction to 50 µL with 20 µL RNase-free water, 5 µL 10X polyA polymerase buffer and 5 µL E. coli polyA polymerase.

SECTION: Part 2: In vitro transcription and polyA tailing
16. Incubate at 37 °C for 30 min.

SECTION: Part 3: Purification of IVT product
17. Purify the polyadenylated mRNA on 50 µg New England Biolabs Monarch RNA cleanup columns (2 of 20 µL reactions per column) (T2040L).

SECTION: Part 3: Purification of IVT product
18. Dilute each 20 µL reaction with 100 µL binding buffer and 150 µL 100% ethanol.

SECTION: Part 3: Purification of IVT product
19. Add the two diluted reactions to one column and spin at 16000 x g, 1 min.

SECTION: Part 3: Purification of IVT product
20. Wash each column with wash buffer and spin twice (see below).
SECTION: Part 3: Purification of IVT product
21. Elute in25 µL of RNase-free H2O per column, incubate for 1 min at 37 Room temperature.

SECTION: Part 3: Purification of IVT product
22. Spin at 16000 x g, 1 min into fresh 1.5 mLtubes and pool elution together into one tube.

SECTION: Part 3: Purification of IVT product
23. Measure the RNA concentration using a Nanodrop1000c spectrophotometer.
 

SECTION: Part 3: Purification of IVT product
24. Store the mRNA at -80 °C and avoid repeated freezing and thawing.

SECTION: Part 3: Purification of IVT product
20.1. Wash with 500 µL wash buffer (spin at 16000 x g, 1 min). (1/2)
SECTION: Part 3: Purification of IVT product
20.2. Wash with 500 µL wash buffer (spin at 16000 x g, 1 min). (2/2)