TITLE: In vitro transcription of guide RNAs and 5&#39;-triphosphate removal
AUTHORS: Mark Dewitt,  Julia Wong, Beeke Wienert, Moritz F Schlapansky, Eric Aird

[DESCRIPTION]
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. 



[STEPS]
SECTION: Design sgRNA and order PCR oligos
1. 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’ )

 

SECTION: Generate in vitro transcription DNA template
2. 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).

  
SECTION: In vitro transcription DNA template PCR
3. 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
SECTION: In vitro T7 transcription
4. 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
   volume reagent   2 µl 10x Buffer 1x   2 µl ATP (100 mM), 10 mM   2 µl GTP (100 mM), 10 mM   2 µl CTP (100 mM), 10 mM   2 µl UTP (100 mM), 10 mM   8 µl DNA template from Step 3   2 µl T7 RNA polymerase mix   
 
 
SECTION: In vitro T7 transcription
4.1. Incubate transcription mix for ~18 hours (over night) at 37˚ in a thermocycler
1080 min
37 10
SECTION: DNA template removal
5. Remove DNA template by adding 1 µl of RNase-free DNase; incubate 15 min at 37C in thermocycler
15 min
 
37 10
SECTION: sgRNA purification
6. 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:

  
 
SECTION: sgRNA purification
6.1. 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
SECTION: sgRNA purification
6.2. Add 450 µl Isopropanol and mix well by pipetting.
 

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

SECTION: sgRNA purification
6.4. Add 700 µl RWT Buffer; Spin for 15 sec at 10.000 g.
Discard the flow-through.
 

SECTION: sgRNA purification
6.5. Add 500 µl RPE Buffer; Spin for 2 min at 10.000 g.

 
SECTION: sgRNA purification
6.6.  Move spin column to a new collection tube and spin for 1 min at 10.000 g to dry the membrane completely.
SECTION: sgRNA purification
6.7. 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. 
SECTION: Removal of 5&#x27;-triphosphate groups
7. 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.
 

SECTION: Removal of 5&#x27;-triphosphate groups
7.1. 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

   
7.2. Mix well and incubate at 37C for 3h
180 min
37 10
SECTION: 2nd sgRNA purification
8. 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:

  
 
SECTION: 2nd sgRNA purification
8.1. 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
SECTION: 2nd sgRNA purification
8.2. Add 450 µl Isopropanol and mix well by pipetting.
 

SECTION: 2nd sgRNA purification
8.3. Transfer sample (~900 µl) to an RNeasy mini spin column; spin for 15 sec at 10.000 g.
Discard the flow-through.
 

SECTION: 2nd sgRNA purification
8.4. Add 700 µl RWT Buffer; Spin for 15 sec at 10.000 g.
Discard the flow-through.
 

SECTION: 2nd sgRNA purification
8.5. Add 500 µl RPE Buffer; Spin for 2 min at 10.000 g.

 
SECTION: 2nd sgRNA purification
8.6.  Move spin column to a new collection tube and spin for 1 min at 10.000 g to dry the membrane completely.
SECTION: 2nd sgRNA purification
8.7. 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. 
SECTION: sgRNA measurement/QC
9. 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
7.3. 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.