In vitro synthesis of PE2 (nCas9-MMLV RT fusion) polyA mRNA using T7 RNA polymerase

Donald  Rio

Sep 06, 2022

In vitro synthesis of PE2 (nCas9-MMLV RT fusion) polyA mRNA using T7 RNA polymerase

DOI

dx.doi.org/10.17504/protocols.io.b3fmqjk6

Donald Rio¹

¹Dept. of Molecular and Cell Biology, University of California, Berkeley

Donald Rio: don_rio@berkeley.edu

Ezgi Booth

University of California, Berkeley

DOI: dx.doi.org/10.17504/protocols.io.b3fmqjk6

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

https://doi.org/10.7554/eLife.79208

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: May 31, 2024

Protocol Integer ID: 56525

Keywords: Linearization of plasmid, In vitro transcription, Purification of IVT product, polyA tailing, ASAPCRN

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 
ReagentHiScribe T7 ARCA mRNA Kit (with Tailing) - 20 rxnsNew England BiolabsCatalog #E2060S 
 ReagentMonarch RNA Cleanup Kit (50 μg) - 100 prepsNew England BiolabsCatalog #T2040L 
 ReagentPmeI - 2,500 unitsNew England BiolabsCatalog #R0560L  
ReagentDNase I (RNase-free) - 5,000 unitsNew England BiolabsCatalog #M0303L 
ReagentE.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

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 Amount100 µg   plasmid with 200 units Pme I restriction endonuclease in a Amount1 mL   reaction using CutSmart buffer forDuration04:00:00  . at Temperature37 °C  .

Purify the cleaved DNA by phenol-chloroform extraction, collect the upper (aqueous) phase and transfer to fresh Amount1.5 mL   Eppendorf tubes.

Add Concentration5 Molarity (M)   NaCl to a final concentration of Concentration0.1 Molarity (M)   (Amount20 µL  ) and 3 volumes of 100% ethanol.

Leave atTemperature-80 °C   for  Duration02:00:00   to DurationOvernight   to precipitate.

Spin Centrifigation16000 x g, 4°C, 00:10:00 .

10m

Remove the ethanol and air dry forDuration00:30:00  .

30m

Resuspend at Amount500 µL   in TE (Concentration10 millimolar (mM)   Tris-HCl, Ph7.5 , Concentration1 millimolar (mM)  EDTA) buffer.  Store the DNA sample atTemperature-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 Amount20 µL   in vitro transcription reactions using Amount1 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 Temperature37 °C   for Duration02:00:00   in an incubator (not a temp block).

For each Amount20 µL   reaction, add Amount2 µL  DNase I (stock isAmount2 U/uL  ).

Incubate at Temperature37 °C   for Duration00:15:00  .

15m

Dilute the reaction to Amount50 µL   with Amount20 µL   RNase-free water, Amount5 µL   10X polyA polymerase buffer and Amount5 µL   E. coli polyA polymerase.

Incubate at Temperature37 °C   for Duration00:30:00  .

30m

Part 3: Purification of IVT product

Purify the polyadenylated mRNA on Amount50 µg   New England Biolabs Monarch RNA cleanup columns (2 of Amount20 µL   reactions per column) (T2040L).

Dilute each Amount20 µL   reaction with Amount100 µL   binding buffer and Amount150 µL   100% ethanol.

Add the two diluted reactions to one column and spin at Centrifigation16000 x g, 00:01:00 .

Wash each column with wash buffer and spin twice (see below).

Wash with Amount500 µL   wash buffer (spin at Centrifigation16000 x g, 00:01:00 ). (1/2)

Wash with Amount500 µL   wash buffer (spin at Centrifigation16000 x g, 00:01:00 ). (2/2)

Elute inAmount25 µL   of RNase-free H2O per column, incubate for Duration00:01:00   at TemperatureRoom temperature  .

Spin at Centrifigation16000 x g, 00:01:00  into fresh Amount1.5 mL  tubes and pool elution together into one tube.

Measure the RNA concentration using a Nanodrop1000c spectrophotometer.
Note
The yield from the total of 8 reactions is usually ~Amount200 µg  .

Store the mRNA at Temperature-80 °C   and avoid repeated freezing and thawing.

Public workspaceIn vitro synthesis of PE2 (nCas9-MMLV RT fusion) polyA mRNA using T7 RNA polymerase

In vitro synthesis of PE2 (nCas9-MMLV RT fusion) polyA mRNA using T7 RNA polymerase