May 02, 2023

Public workspaceIn vitro transcription of guide RNAs and 5'-triphosphate removal V.17

Nature Communications
DOI
dx.doi.org/10.17504/protocols.io.n2bvjyp5vk5w/v17
  • 1UC BERKELEY-IGI;
  • 2ETHZ - ETH Zurich
Jacob E Corn
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DOI: dx.doi.org/10.17504/protocols.io.n2bvjyp5vk5w/v17
External link: https://doi.org/10.1371/journal.pbio.2005840
Protocol Citation: Mark Dewitt, Julia Wong, Beeke Wienert, Moritz F Schlapansky, Eric Aird 2023. In vitro transcription of guide RNAs and 5'-triphosphate removal. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjyp5vk5w/v17Version created by Jacob E Corn
Manuscript citation:
Wienert B, Shin J, Zelin E, Pestal K, Corn JE (2018) In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway. PLoS Biol 16(7): e2005840. doi: 10.1371/journal.pbio.2005840
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: May 02, 2023
Last Modified: May 02, 2023
Protocol Integer ID: 81273
Abstract
sgRNA template assembly, in vitro T7 transcription, and sgRNA column cleanup to remove 5'-triphosphate groups
Guidelines
The primers used are: one long, variable oligo that carries the T7 promoter and desired guide sequence; an 82-nt constant oligo that carries the 3' end of the sgRNA; two short external primers for amplification.
Assembly Oligos:
T7FwdVarV2 oligo (5’-TAATACGACTCACTATAG--protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ )
T7RevLongV2 oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ )

Amplification Primers:
T7FwdAmpV2 primer (5’-TAATACGACTCACTATAG-3’)
T7RevAmpV2 primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )

Designing the guide sequence:The T7 transcript starts with the G before the dashes in the Fwd assembly oligo. If your target calls for a G at the 5’ end of the guide, use the one already included in T7FwdVar design. If your target calls for a different base at the 5’ end, put the whole guide sequence in T7FwdVar; there will be an extra G on the end that should have minimal effect, unless your guide is quite short.
Making the template: we have used the Phusion-HF DNA polymerase. Although the amplification primers have quite different Tm’s, they work together fine. 


Materials
STEP MATERIALS
ReagentDNase I (RNase-free) - 1,000 unitsNew England BiolabsCatalog #M0303S
ReagentHiScribe T7 High Yield RNA Synthesis Kit - 50 rxnsNew England BiolabsCatalog #E2040S
ReagentPhusion High-Fidelity PCR Kit - 50 rxns (50 ul vol)New England BiolabsCatalog #E0553S
ReagentmiRNeasy mini KitCatalog #217004
ReagentQuick CIP New England BiolabsCatalog #M0525
Protocol materials
ReagentmiRNeasy mini KitCatalog #217004
In Materials and 2 steps
ReagentQuick CIP New England BiolabsCatalog #M0525
Materials, Step 7.1
ReagentDNase I (RNase-free) - 1,000 unitsNew England BiolabsCatalog #M0303S
Materials, Step 5
ReagentHiScribe T7 High Yield RNA Synthesis Kit - 50 rxnsNew England BiolabsCatalog #E2040S
Materials, Step 4
ReagentPhusion High-Fidelity PCR Kit - 50 rxns (50 ul vol)New England BiolabsCatalog #E0553S
Materials, Step 2
Design sgRNA and order PCR oligos
Design sgRNA and order PCR oligos
Add the desired protospacer sequence to the T7FwdVarV2 oligo and order the oligo from your favorite oligonucleotide supplier. There are many programs available for protospacer design that attempt to optimize on- and/or off-target activity. Which program is most useful depends upon many factors including type of editing, organism being edited, etc. Choice of protospacer design program is beyond the scope of this protocol.
The transcript will start with the bolded G just 5’ of the dashes in the T7FwdVarV2 oligo. T7 RNA polymerase requires a 5’ G for proper transcript initiation. If your protospacer has a G at the 5’ end, you can omit it from the T7FwdVarV2 design to avoid duplication of the G. If your protospacer has a C, T, or A at the 5’ end, add the whole protospacer sequence to T7FwdVarV2. In this case, there will be an extra G added to the 5’ end of the protospacer, but literature indicates this will have minimal effect unless your guide is very short.
Primers: 
T7FwdVarV2 oligo (5’-TAATACGACTCACTATAG--protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ )
T7RevLongV2 oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ )
T7FwdAmpV2 primer (5’-TAATACGACTCACTATAG-3’)
T7RevAmpV2 primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )

Note
These V2 oligos differ from a previous version due to reported benefits using modified sgRNA structures that might enhance binding to Cas9 or increase sgRNA stability (see https://doi.org/10.1186/s13059-015-0846-3).

Generate in vitro transcription DNA template
Generate in vitro transcription DNA template
For each T7FwdVarV2 oligo you designed, set up the following PCR (total volume should be 20.0 µL). Make sure everything is RNase free and filter tips are used. Furthermore, wipe down everything (in every step of protocol) with RNase Away to ensure no contamination with RNAse. 
10.6 µl DEPC-treated H2O
4.0 uL 5x Phusion HF Buffer                                       
0.4 µl 10 mM dNTPs                     
0.4 µl T7FwdVarV2 (1 µM)     
0.4 µl T7RevLongV2 (1 µM)    
2 µl T7FwdAmpV2 (10 µM)             
2 µl T7RevAmpV2 (10 µM)              
0.2 µl Phusion HF DNA polymerase (2u/µl)
If making multiple sgRNA templates, prepare a master mix with all components except T7FwdVarV2. Include a no template control (omit T7FwdVarV2).

ReagentPhusion High-Fidelity PCR Kit - 50 rxns (50 ul vol)New England BiolabsCatalog #E0553S
In vitro transcription DNA template PCR
In vitro transcription DNA template PCR
Run PCR:
98˚ 30 sec
98˚ 10 sec
51˚ 10 sec  
72˚ 10 sec
      30x steps 2-4
72˚ 2 min
4˚ hold


No PCR cleanup necessary at this point
In vitro T7 transcription
In vitro T7 transcription
We like to use HiScribe T7 High Yield RNA Synthesis Kit but any T7 RNA synthesis kit should be fine.
Mix the following to make 20 µl total T7 transcription mix
AB
volumereagent
2 µl10x Buffer 1x
2 µlATP (100 mM), 10 mM
2 µlGTP (100 mM), 10 mM
2 µlCTP (100 mM), 10 mM
2 µlUTP (100 mM), 10 mM
8 µlDNA template from Step 3, (usually ~4µg, 50 pmol)
2 µlT7 RNA polymerase mix
ReagentHiScribe T7 High Yield RNA Synthesis Kit - 50 rxnsNew England BiolabsCatalog #E2040S
Incubate transcription mix for ~18 hours (over night) at 37˚ in a thermocycler
Duration18:00:00
Temperature37 °C
DNA template removal
DNA template removal
Remove DNA template by adding 1 µl of RNase-free DNase; incubate 15 min at 37C in thermocycler
Duration00:15:00 37C incubation
ReagentDNase I (RNase-free) - 1,000 unitsNew England BiolabsCatalog #M0303S
Temperature37 °C
sgRNA purification
sgRNA purification
sgRNAs need to be purfied before dephosphorylation. There are different methods one could purify their sgRNAs. We therefore tested different purification kits and found that the Qiagen miRNeasy Tissue/Cells Advanced Mini Kit gives the most consistent and highest yields. To purify our sgRNAs with this kit, we follow the manufacturer's instructions with the following modifications:

ReagentmiRNeasy mini KitCatalog #217004
Note
Yields of from column purification using the Qiagen RNeasy Mini kit can be low as the sgRNAs are small and most kits are designed to bind longer RNAs. Our yields are consistently higher when using the miRNeasy Tissue/Cells Advanced Mini Kit from Qiagen.
Adjust sgRNA sample to a volume of 100 µl with RNase-free water.
Add 350 µl RLT Buffer to the sample and mix well by pipetting
Add 450 µl Isopropanol and mix well by pipetting.

Transfer sample (~900 µl) to an RNeasy mini spin column; spin for 15 sec at 10.000 g.
Discard the flow-through.

Add 700 µl RWT Buffer; Spin for 15 sec at 10.000 g.
Discard the flow-through.
Add 500 µl RPE Buffer; Spin for 2 min at 10.000 g.
Move spin column to a new collection tube and spin for 1 min at 10.000 g to dry the membrane completely.
Move spin column to an RNAse-free 1.5 ml microfuge tube
Add 33 µl DEPC-treated H2O; spin 1 min
Optional: Repeat the elution to collect any remaining RNA on the column and increase RNA concentration.
Removal of 5'-triphosphate groups
Removal of 5'-triphosphate groups
1d 6h
1d 6h
T7 in vitro transcription results in RNA carrying a 5'-triphosphate group. This triggers a RIG-I-mediated innate immnue response in mammalian cells and can cause cell death, particularly in primary cells. Depending on your target cells, we recommend treating your IVT sgRNA with a heat-labile version of calf intestinal alkaline phosphatase (Quick CIP) before proceeding with editing. We found that Quick CIP treatment must be rigorous to completely remove all 5-PPP groups from your RNA, hence the initial purification prior to dephosphorylation. However, Quick CIP binds tightly to RNA and NEB recommends to only use the minimal amount needed.
Note
Removal of the 5'-triphosphate group is not always necessary. We prefer the removal when working with primary cells.

For a 40µl reaction:
Add 4 ul of NEB CutSmart Buffer (comes with the Quick CIP enzyme)
Add 2 ul (10 units) of Quick CIP
Add X µl of H2O to a final reaction volume of 40 µl

ReagentQuick CIP New England BiolabsCatalog #M0525
Mix well and incubate at 37C for 3h
Duration03:00:00
Temperature37 °C
3h
A cell-based IFN stimulation assay can be used to confirm dephosphorylation as performed in https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2005840. This is helpful when making your first gRNAs, but typically not something that we do every time.
2nd sgRNA purification
2nd sgRNA purification
1d 6h
1d 6h
sgRNAs need to be purified before editing. There are different methods one could purify their sgRNAs. We therefore tested different purification kits and found that the Qiagen miRNeasy Tissue/Cells Advanced Mini Kit gives the most consistent and highest yields. To purify our sgRNAs with this kit, we follow the manufacturer's instructions with the following modifications:

ReagentmiRNeasy mini KitCatalog #217004
Note
Yields of from column purification using the Qiagen RNeasy Mini kit can be low as the sgRNAs are small and most kits are designed to bind longer RNAs. Our yields are consistently higher when using the miRNeasy Tissue/Cells Advanced Mini Kit from Qiagen.
Adjust sgRNA sample to a volume of 100 µl with RNase-free water.
Add 350 µl RLT Buffer to the sample and mix well by pipetting
Add 450 µl Isopropanol and mix well by pipetting.

Transfer sample (~900 µl) to an RNeasy mini spin column; spin for 15 sec at 10.000 g.
Discard the flow-through.

Add 700 µl RWT Buffer; Spin for 15 sec at 10.000 g.
Discard the flow-through.
Add 500 µl RPE Buffer; Spin for 2 min at 10.000 g.
Move spin column to a new collection tube and spin for 1 min at 10.000 g to dry the membrane completely.
Move spin column to an RNAse-free 1.5 ml microfuge tube
Add 33 µl DEPC-treated H2O; spin 1 min
Optional: Repeat the elution to collect any remaining RNA on the column and increase RNA concentration.
sgRNA measurement/QC
sgRNA measurement/QC
Measure your RNA concentration by Nanodrop or Qubit. You can also check integrity/corrrect size of the sgRNA(s) on the RNA tapestation. Store sgRNAs at -80 °C