TITLE: In Vitro Transcription for dgRNA
AUTHORS: Amy Lyden, Emily Crawford, Jenai Quan, Saharai Caldera, Lara Pesce-Ares

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For FLASH, DASH, and other CRISPR-cas9 protocols, we use T7 to transcribe our crRNA and tracrRNA to make dgRNA for cas9. It is more time-, labor-, and cost-effective to make dgRNAs instead of sgRNAs for large guide RNA libraries such as those used in DASH or FLASH. The two components of the dual guides are the crRNA (containing your variable 20 nt target plus a 22 nt constant region) and the tracrRNA (a 72 nt constant region). </div></div>

[STEPS]
?. [Annealing T7 to crRNA and tracrRNA template]
Pool your crRNA in equimolar amounts. Usually, we order 96-well plates of 96 crRNA templates from IDT, with the oligos diluted in water at a concentration of 10µM
?. [Annealing T7 to crRNA and tracrRNA template]
Add an equimolar amount of T7 primer to your crRNA pool. For example, reconstitute T7 primer to 10µM, and pool 500µL of your 10µM crRNA pool with 500µL of your T7 primer at 10µM.
?. [In Vitro Transcription]
Make all reagents are at room temperature. Prepare the reaction mixtures below in the order specified. DO NOT prepare the reaction on ice, as some components are prone to precipitation.
?. [In Vitro Transcription]
Prepare tracrRNA mixture by adding the following reagents to a 1.5mL tube in order.   AB1VolumetracrRNA21X3470µLRNAse-free water4120µL10X T7 buffer*5300µLNTPs 25mM each6100µLT7 enzyme (1:100 diluted in 1X T7 buffer, final conc: 100µg/mL)710µL / 8 µgtracrRNA template with T7 annealed, 800ng/µL81mLTOTAL
AB1VolumetracrRNA21X3470µLRNAse-free water4120µL10X T7 buffer*5300µLNTPs 25mM each6100µLT7 enzyme (1:100 diluted in 1X T7 buffer, final conc: 100µg/mL)710µL / 8 µgtracrRNA template with T7 annealed, 800ng/µL81mLTOTAL
?. [In Vitro Transcription]
Prepare crRNA mixture by adding the following reagents to a 1.5mL tube in order.   AB1VolumecrRNA21X3380µLRNAse-free water4120µL10X T7 buffer*5300µLNTPs 25mM each6100µLT7 enzyme (1:100 diluted in 1X T7 buffer, final conc: 100µg/mL)7100µL / 4µgcrRNA template with T7 annealed, 40ng/µL81mLTOTAL
AB1VolumecrRNA21X3380µLRNAse-free water4120µL10X T7 buffer*5300µLNTPs 25mM each6100µLT7 enzyme (1:100 diluted in 1X T7 buffer, final conc: 100µg/mL)7100µL / 4µgcrRNA template with T7 annealed, 40ng/µL81mLTOTAL
?. [Annealing T7 to crRNA and tracrRNA template]
Add an equimolar amount of T7 primer to your tracr RNA. For example, if you have reconstituted your tracrRNA to 100µM, pool 500µL of your tracrRNA at 100µM to 500µL of your T7 primer at 100µM.
?. [Annealing T7 to crRNA and tracrRNA template]
Anneal tracrRNA template + T7 primer and crRNA template + T7 primer by heating to  on a heat block or thermocycler for   and allowing them to cool to room temperature slowly on the bench
?. [Prepare for IVT reaction]
Nanodrop or Qubit your tracrRNA template and crRNA template using ssDNA setting or kit.
?. [Prepare for IVT reaction]
Dilute your tracrRNA template with T7 primer annealed to 800ng/µL with water.  Dilute your crRNA template with T7 primers to 40ng/µL with water.
Can store these as aliquots at
?. [In Vitro Transcription]
Mix NTPs together in equimolar amounts to have enough for the following reactions. For example, mix 200µL A NTP at 100mM, 200µL C NTP at 100mM, 200µL G NTP at 100mM, and 200µL U NTP at 100mM for a final solution of 25mM each.
?. [In Vitro Transcription]
Prepare a small amount of 1X T7 buffer (200µL for the reaction below). Dilute your T7 enzyme to 100µg/mL in 1X T7 buffer.
?. [In Vitro Transcription]
Incubate at for  and proceed immediately to purification.
?. [RNA Purification with SPRI beads]
Use homemade SPRI beads or Ampure beads to purify gRNAs after transcription.
Note: Alternatively, RNA may be purified using a Zymo RNA Clean & Concentrator-5 or -25 Kit (Zymo Research R1015)
?. [RNA Purification with SPRI beads]
Equilibrate SPRI beads to room temperature.
?. [RNA Purification with SPRI beads]
For every 200 µL of IVT reaction, add 300µL of 100% ethanol. The solution should turn a cloudy white (precipitation of RNA) upon addition of ethanol. This step helps the short RNAs bind to the SPRI beads. This can be done in a 15mL tube or several 1.5mL tubes.
?. [RNA Purification with SPRI beads]
For every 500µL of IVT reaction + 100% ethanol, add 500µL of SPRI beads to the solution of IVT reaction and ethanol and mix well by inverting or pipetting with a P1000.
?. [RNA Purification with SPRI beads]
Incubate at room temperature for .
?. [RNA Purification with SPRI beads]
Divide this mixture up into an appropriate number of 1.5mL Lo-Bind Eppendorf tubes and place on a 1.5 mL magnetic separation rack OR use a 15mL magnetic tube rack.
?. [RNA Purification with SPRI beads]
Wait to allow the beads to separate if using a 1.5mL rack OR  if using a 15mL rack.
?. [RNA Purification with SPRI beads]
Remove and discard the supernatant.
?. [RNA Purification with SPRI beads]
Rinse the beads with 1mL of 80% ethanol if using a 1.5mL tube or ~10mL of 80% ethanol if using the 15mL tube. It is not necessary to resuspend the beads.
?. [RNA Purification with SPRI beads]
Wait  then remove and discard the ethanol.
?. [RNA Purification with SPRI beads]
Repeat the wash step as described above. (Add the same amount of 80% ethanol, wait , then discard the ethanol.)
?. [RNA Purification with SPRI beads]
Remove residual ethanol that collects at the bottom of the tube by using a P200 or P20.
?. [RNA Purification with SPRI beads]
Air dry the beads for in a 1.5mL tube or  in a 15mL tube, or until the beads lose their glossy appearance. Sufficiently dry beads will appear matte. Be careful not to let the beads get too dry (appearing cracked or dusty).
?. [RNA Purification with SPRI beads]
Elute the RNA by resuspending the beads with an appropriate amount of nuclease-free H2O depending on the desired volume and concentration. For DASH guides which need to be at a final concentration of 40µM, lower is better (80µL per 1X reaction). For FLASH guides which need to be at a final concentration of 4µM, a higher volume can be used.
?. [RNA Purification with SPRI beads]
Allow the RNA to elute off the beads by incubating at room temperature for .
?. [RNA Purification with SPRI beads]
If necessary, pulse-spin the tubes to collect any liquid along the sides of the tubes.
?. [RNA Purification with SPRI beads]
Place the tubes on the magnetic rack and allow them to separate until water is clear. This will take 2-5 minutes for a 1.5mL tube, 5-10 minutes for a 15mL tube.
?. [RNA Purification with SPRI beads]
Collect the eluted RNA, being careful not to take up beads. (Eg. If eluted in 80uL, collect 75µL).
?. [Quantify, anneal and aliquot dgRNA]
Using the HS RNA Qubit kit, quantify 1µL of the eluted tracrRNA and 1µL of the eluted crRNA. Follow standard HS RNA Qubit protocol.
?. [Quantify, anneal and aliquot dgRNA]
crRNA: For DASH, dilute the stock crRNA to 1100 ng/μL. This is equivalent to 80 μM. For FLASH or other lower concentration needs, dilute to 110 ng/µL or 8µM
?. [Quantify, anneal and aliquot dgRNA]
tracrRNA: For DASH, dilute the stock crRNA to 1900 ng/μL. This is equivalent to 80 μM. For FLASH or other lower concentration needs, dilute to 190 ng/µL or 8µM
?. [Quantify, anneal and aliquot dgRNA]
If you want to check purity, run your 1:100 dilutions on a small RNA chip on the bioanalyzer immediately after denaturing them by heating to  for . The crRNA and tracrRNA are 42 nt and 72 nt long, respectively.
?. [Quantify, anneal and aliquot dgRNA]
To form the dgRNA complex, mix together equilmolar amounts of crRNA and tracrRNA (equal volumes of the 80 μM crRNA stock and the 80 μM tracrRNA stock), heat to  for  and then cool slowly on the bench.
?. [Quantify, anneal and aliquot dgRNA]
Store dgRNA at  in small aliquots in order to avoid freeze-thaws. If there is crRNA or tracrRNA left over, freeze them separately -- the dgRNA complex can be formed in the same way ( for ) immediately prior to complexing with Cas9.