Sep 07, 2022
In vitro synthesis of PE2 (nCas9-MMLV RT fusion) polyA mRNA using T7 RNA polymerase
- Donald Rio1
- 1Dept. of Molecular and Cell Biology, University of California, Berkeley
- Donald Rio: [email protected]
External link: https://doi.org/10.7554/eLife.79208
Protocol Citation: Donald Rio 2022. In vitro synthesis of PE2 (nCas9-MMLV RT fusion) polyA mRNA using T7 RNA polymerase. protocols.io https://dx.doi.org/10.17504/protocols.io.b3fmqjk6
Manuscript citation:
Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner (2022) Highly efficient generation of isogenic pluripotent stem cell models using prime editing eLife 11:e79208
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 04, 2022
Last Modified: June 01, 2024
Protocol Integer ID: 56525
Keywords: Linearization of plasmid, In vitro transcription, Purification of IVT product, polyA tailing, ASAPCRN, using t7 rna polymerase, reverse transcriptase fusion protein, t7 rna polymerase, genome editing, polyadenylated messenger rna, use in genome editing, messenger rna, prime editing, called prime editing, mrna, synthesis of pe2, cas9 nickase, synthesis, genome, mmlv rt fusion, ncas9
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
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.
Attachments
336-739.docx
18KB
Materials
Materials
HiScribe T7 ARCA mRNA Kit (with Tailing) - 20 rxnsNew England BiolabsCatalog #E2060S
Monarch RNA Cleanup Kit (50 μg) - 100 prepsNew England BiolabsCatalog #T2040L
PmeI - 2,500 unitsNew England BiolabsCatalog #R0560L
DNase I (RNase-free) - 5,000 unitsNew England BiolabsCatalog #M0303L
E.coli Poly (A) Polymerase - 500 unitsNew England BiolabsCatalog #M0276L
- CutSmart buffer
- 5M NaCl
- 100% ethanol
- 10mM Tris-HCl
- 1mM EDTA
- TE buffer
- New England Biolabs Monarch RNA cleanup column
- Nanodrop1000c spectrophotometer
Troubleshooting
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 100 µg plasmid with 200 units Pme I restriction endonuclease in a 1 mL reaction using CutSmart buffer for04:00:00 . at 37 °C .
4h
Purify the cleaved DNA by phenol-chloroform extraction, collect the upper (aqueous) phase and transfer to fresh 1.5 mL Eppendorf tubes.
Add 5 Molarity (M) NaCl to a final concentration of 0.1 Molarity (M) (20 µL ) and 3 volumes of 100% ethanol.
Leave at-80 °C for 02:00:00 to Overnight to precipitate.
4h
Spin 16000 x g, 4°C, 00:10:00 .
10m
Remove the ethanol and air dry for00:30:00 .
30m
Resuspend at 500 µL in TE (10 millimolar (mM) Tris-HCl, 7.5 , 1 millimolar (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 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.
Incubate at 37 °C for 02:00:00 in an incubator (not a temp block).
2h
For each 20 µL reaction, add 2 µL DNase I (stock is2 U/uL ).
Incubate at 37 °C for 00:15:00 .
15m
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.
Incubate at 37 °C for 00:30:00 .
30m
Part 3: Purification of IVT product
5m
Purify the polyadenylated mRNA on 50 µg New England Biolabs Monarch RNA cleanup columns (2 of 20 µL reactions per column) (T2040L).
Dilute each 20 µL reaction with 100 µL binding buffer and 150 µL 100% ethanol.
Add the two diluted reactions to one column and spin at 16000 x g, 00:01:00 .
1m
Wash each column with wash buffer and spin twice (see below).
Wash with 500 µL wash buffer (spin at 16000 x g, 00:01:00 ). (1/2)
1m
Wash with 500 µL wash buffer (spin at 16000 x g, 00:01:00 ). (2/2)
1m
Elute in25 µL of RNase-free H2O per column, incubate for 00:01:00 at Room temperature .
1m
Spin at 16000 x g, 00:01:00 into fresh 1.5 mL tubes and pool elution together into one tube.
1m
Measure the RNA concentration using a Nanodrop1000c spectrophotometer.
Note
The yield from the total of 8 reactions is usually ~200 µg .
Store the mRNA at -80 °C and avoid repeated freezing and thawing.