In vitro transcription of guide RNAs and 5'-triphosphate removal

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’-TAATACGACTCACTATA G --protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ )

T7RevLongV2 oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ )

T7FwdAmpV2 primer (5’-TAATACGACTCACTATAG-3’)

T7RevAmpV2 primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )

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).

Primers: 

T7FwdVarV2 oligo (5’-TAATACGACTCACTATA G --protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ )

T7RevLongV2 oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ )

T7FwdAmpV2 primer (5’-TAATACGACTCACTATAG-3’)

T7RevAmpV2 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 total T7 transcription mix

A	B
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

18h 0m 0s

37

Remove DNA template by adding 1 µl of RNase-free DNase; incubate 15 min at 37C in thermocycler

0h 15m 0s

37

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 a heat-labile version of calf intestinal alkaline phosphatase (Quick CIP) before proceeding to the purification step. We found that Quick CIP treatment must be rigorous to completely remove all 5-PPP groups from your RNA. However, Quick CIP binds tightly to RNA and NEB recommends to only use the minimal amount needed.

Add 3ul of NEB CutSmart Buffer (comes with the Quick CIP enzyme)

Add 4µl of H2O

Add 2ul (2 units) of Quick CIP

Mix well and incubate at 37C for 3h

3h 0m 0s

37

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:

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 transfer 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