TITLE: In vitro transcription of guide RNAs and 5'-triphosphate removal
AUTHORS: Mark Dewitt,  Julia Wong, Beeke Wienert, Moritz Schlapansky

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">sgRNA template assembly, in vitro T7 transcription, and sgRNA column cleanup to remove 5'-triphosphate groups</div></div>

[STEPS]
?. [Design sgRNA and order PCR oligos.]
Add the desired protospacer sequence to the T7FwdVar 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 T7FwdVar 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 T7FwdVar 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 T7FwdVar. 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: T7FwdVar oligo (5’-TAATACGACTCACTATAG--protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ )T7RevLong oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ )T7FwdAmp primer (5’-TAATACGACTCACTATAG-3’)T7RevAmp primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )
?. [Generate in vitro transcription DNA template]
For each T7FwdVar 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 T7FwdVar (1 µM)       0.4 µl T7RevLong (1 µM)      2 µl T7FwdAmp (10 µM)               2 µl T7RevAmp (10 µM)                0.2 µl Phusion HF DNA polymerase (2u/µl)If making multiple sgRNA templates, prepare a master mix with all components except T7FwdVar. Include a no template control (omit T7FwdVar).Primers: T7FwdVar oligo (5’-TAATACGACTCACTATAG--protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ )T7RevLong oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ )T7FwdAmp primer (5’-TAATACGACTCACTATAG-3’)T7RevAmp primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )
?. [In vitro transcription DNA template PCR]
Run PCR:98˚ 30 sec98˚ 10 sec 51˚ 10 sec  72˚ 10 sec      30x steps 2-472˚ 2 min4˚ holdNo PCR cleanup necessary at this point
?. [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  AB1volumereagent22 µl10x Buffer 1x32 µlATP (100 mM), 10 mM42 µlGTP (100 mM), 10 mM52 µlCTP (100 mM), 10 mM62 µlUTP (100 mM), 10 mM78 µlDNA template from Step 3, (usually ~4µg, 50 pmol)
82 µlT7 RNA polymerase mix
AB1volumereagent22 µl10x Buffer 1x32 µlATP (100 mM), 10 mM42 µlGTP (100 mM), 10 mM52 µlCTP (100 mM), 10 mM62 µlUTP (100 mM), 10 mM78 µlDNA template from Step 3, (usually ~4µg, 50 pmol)
82 µlT7 RNA polymerase mix
?. [In vitro T7 transcription]
Incubate transcription mix for ~18 hours (over night) at 37˚ in a thermocycler
37 °C
?. [DNA template removal]
Remove DNA template by adding 1 µl of RNase-free DNase; incubate 15 min at 37C in thermocycler
[37C incubation]
37 °C
?. [Removal of 5'-triphosphate groups]
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. We highly recommend treating your IVT sgRNA with recombinant Shrimp Alkaline Phosphatase (rSAP) before proceeding to the purification step. We found that rSAP treatment must be rigorous to completely remove all 5-PPP groups from your RNA. However, rSAP binds tightly to RNA and NEB recommends to only use the minimal amount needed.
?. [Removal of 5'-triphosphate groups]
Add 3ul of NEB CutSmart Buffer (comes with the rSAP enzyme)Add 4µl of H2OAdd 2ul (2 units) of rSAP
?. [Removal of 5'-triphosphate groups]
Mix well and incubate at 37C for 3h
37 °C
?. [sgRNA purification]
sgRNAs need to be purfied before transfection. There are different methods one could purify their sgRNAs. We found that while SPRI bead clean-up of RNAs is quick and gives reliable yields (see older versions of protocol), SPRI bead purified sgRNAs still cause an elevated immune response even after Phosphatase treatment. We therefore tested different column purification kits and found that column purification of sgRNAs completely eliminates the immune response after Phosphatase treatment.We use the Qiagen RNeasy Mini Prep Kit and follow the manufacturer's instructions with the following modifications:
Yields of from column purification can be low as the sgRNAs are small and most kits are designed to bind longer RNAs. We found the Qiagen RNeasy kit works ok, but others recommend the miRNeasy kit from Qiagen. It might be worth trying a few different kits to optimize your yield.
?. [sgRNA purification]
Adjust sgRNA sample to a volume of 100 µl with RNase-free water.Add 350 µl RLT Buffer to the sample
?. [sgRNA purification]
Add 250 µl 100% ethanol and mix well by pipetting.
?. [sgRNA purification]
Tranfer the sample (~700 µl) to an RNeasy mini spin column; spin for 15 sec at >8000 g  Then transferr the remainder onto the same column; spin for 15 sec
?. [sgRNA purification]
Add 500 µl RPE Buffer; spin 15 sec Repeated this wash step
?. [sgRNA purification]
Moved spin column to a new collection tube and spin for 1 min to dry the membrane completely
?. [sgRNA purification]
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
?. [sgRNA purification]
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 -80C