Editing MRNA Products (section)

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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’ )


For eachT7FwdVar 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’ )


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

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 totalT7 transcriptionmix
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, (usually ~4µg, 50 pmol)"
2 µl	T7 RNA polymerase mix

Incubate transcription mix for ~18 hours (over night) at 37˚ in a thermocycler
18:00:00 START
37 °C

Remove DNA template by adding 1 µl of RNase-free DNase; incubate 15 min at 37C in thermocycler
00:15:00 37C incubationSTART
DNase I (RNase-free) - 1,000 unitsNew England BiolabsCatalog #M0303S
37 °C

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.

Add 3ul of NEB CutSmart Buffer (comes with the rSAP enzyme)
Add 4µl of H2O
Add 2ul (2 units) of rSAP

Shrimp Alkaline Phosphatase (rSAP) - 500 unitsNew England BiolabsCatalog #M0371S

Mix well and incubate at 37C for 3h
03:00:00 START
37 °C

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. 

Adjust sgRNA sample to a volume of 100 µl with RNase-free water.
Add 350 µl RLT Buffer to the sample 

Add 250 µl 100% ethanol and mix well by pipetting.

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
 
Add 500 µl RPE Buffer; spin 15 sec
 Repeated this wash step
 Moved spin column to a new collection tube and spin for 1 min 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


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
https://www.protocols.io/run/in-vitro-transcription-of-guide-rnas-and-5-triphos-bqjbmuin