TITLE: In vitro transcription of crRNA and tracrRNA from DNA oligos for cas9 enrichment and nanopore sequencing (for Bac - PULCE)    
AUTHORS: Olin Silander

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol we describe the steps for the design and production of dual guide RNAs (dgRNAs) from DNA oligos for the enrichment of specific sequences from mixed samples. These enriched molecules can then be sequenced using long read Oxford Nanopore Sequencing. The method as outlined and applied here is termed Bac-PULCE (Bacterial strain and antimicrobial resistance Profiling Using Long reads via CRISPR Enrichment) but can be applied to enrich specific DNA molecules from any samples for downstream ONT sequencing.</div><div class = "text-block">For CRISPR-cas9 protocols including Bac-PULCE, we use T7 polymerase to transcribe the crRNA and tracrRNA to make dgRNA for Cas9. The two components of the dual guides are the crRNA (containing your variable 20 bp target plus a 22 bp constant region) and the tracrRNA (a 72 bp constant region). </div><div class = "text-block">The protocol outlined here is a combination of the two protocols below: </div><div class = "text-block"><a href="https://www.protocols.io/view/in-vitro-transcription-for-dgrna-3bpgimn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/in-vitro-transcription-for-dgrna-3bpgimn</span></a><span> /</span><a href="https://dx.doi.org/10.17504/protocols.io.3bpgimn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">dx.doi.org/10.17504/protocols.io.3bpgimn</span></a></div><div class = "text-block"><a href="https://international.neb.com/protocols/2013/04/02/standard-rna-synthesis-e2050" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://international.neb.com/protocols/2013/04/02/standard-rna-synthesis-e2050</span></a></div><div class = "text-block">And the end product of this protocol is used for Nanopore protocol Cas9-mediated PCR-free enrichment as outlined by Oxford Nanopore - please refer to this protocol in particular for further important detail around sequencing the DNA library. </div><div class = "text-block"><span>The aim of this protocol is to design and produce crRNAs and tracRNAs from DNA oligos, and combine these with Cas9 to cut specific DNA sequences. We will add a T7 RNA polymerase binding site at the 5’ end of the crRNA and tracRNA to allow </span><span style = "font-style:italic;">in vitro</span><span> transcription, as well as a 3’ tracrRNA binding site to the crRNA. To allow crRNA and tracRNA production from DNA oligos, we order our DNA oligos of the reverse complement (see below for more details).</span></div></div>

[STEPS]
?. [Preparation ]
RNAse Zap equipment, gloves and bench prior to starting protocol. Turn on heat block to reach a temperature of  .
95 °C
?. [Annealing T7 to crRNA and tracrRNA template]
Pool your crRNA DNA oligo sequences in equimolar amounts. For small numbers of crRNAs we order templates from IDT with oligos diluted in nuclease free water at a concentration of 10µM. For larger numbers of crRNAs, order in 96-well plate format.
?. [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 125µL of your 10µM crRNA pool with 125µL of your T7 primer at 10µM.
?. [Annealing T7 to crRNA and tracrRNA template]
Add an equimolar amount of T7 primer to your tracrRNA. For example, if you have reconstituted your tracrRNA to 100µM, pool 125µL of your tracrRNA at 100µM to 125µL 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 in a tube rack.
95 °C
?. [In Vitro Transcription]
For IVT we used the T7 E2050S kit from NEB and followed the standard RNA synthesis method. Thaw and keep on ice the necessary components - NTP buffer mix.
?. [In Vitro Transcription]
Assemble the reactions in the following order in nuclease free 1.5ml lobbind tubes for both the crRNA & tracrRNA . total volume can be scaled accordingly. We usually had a total reaction volume of 90µL. 1xCrRNATracrRNANuclease free waterXµLXµLNTP buffer mix30µ30µ Oligo RNA template(1µg)(1µg)T7 Polymerase Mix6µL6µLTotal Volume Mix90µL90µL
1xCrRNATracrRNANuclease free waterXµLXµLNTP buffer mix30µ30µ Oligo RNA template(1µg)(1µg)T7 Polymerase Mix6µL6µLTotal Volume Mix90µL90µL
?. [In Vitro Transcription]
Incubate at overnight. We usually incubate for 16 hours using a heat block.
37 °C
?. [RNA Purification with SPRI beads]
Proceed to purification. We used Ampure beads to purify gRNAs after transcription.
?. [RNA Purification with SPRI beads]
Equilibrate Ampure XP beads to room temperature.
?. [RNA Purification with SPRI beads]
For every 200 µL of the IVT reaction, add 300µL of 100% ethanol (1:1.5 ratio e.g. for 90 µl IVT reaction mixture then add 135 µl of ethanol). The solution should turn a cloudy white (precipitation of RNA) upon addition of ethanol. This step helps the short RNAs bind to the beads. This can be done in several 1.5mL tubes.
?. [RNA Purification with SPRI beads]
For every 500µL of IVT reaction + 100% ethanol, add 500µL of Ampure XP 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]
Place the tubes on a 1.5 mL magnetic separation rack.
?. [RNA Purification with SPRI beads]
Wait to allow the beads to separate if using a 1.5mL rack. Allow a longer time if necessary to ensure proper separation of beads liquid before proceeding. While waiting make up a batch of 80% ethanol for next step.
?. [RNA Purification with SPRI beads]
Make up a batch of 80% ethanol for following wash steps. Usually 5ml is sufficient for washing both crRNA & tracrRNA tubes.
?. [RNA Purification with SPRI beads]
Remove and discard the supernatant while tube remains in magnetic rack.
?. [RNA Purification with SPRI beads]
Rinse the beads with 1mL of 80% ethanol if using a 1.5mL 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 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 the proceeding nanopore sequencing we usually elute with 30-50µL nuclease free H20.
?. [RNA Purification with SPRI beads]
Flick the tubes to combine and 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 5-10 minutes for a 1.5mL tube.
?. [RNA Purification with SPRI beads]
Collect the eluted RNA, being careful not to take up beads.
?. [Quantify, anneal and aliquot dgRNA]
Using the XR RNA Qubit kit, quantify 1µL of the eluted tracrRNA and 1µL of the eluted crRNA. Follow standard XR RNA Qubit protocol. if concentration is too large to read initially make a 1 in 10 dilution.
?. [Quantify, anneal and aliquot dgRNA]
Store RNA at  in small aliquots (we usually store 3µl-6µl aliquots in PCR tubes) in order to avoid freeze-thaws. The crRNA & tracrRNA should be frozen separately. Annealing to make the dgRNAs should be done only prior to sequencing.
-80 °C
?. [Preparing input DNA *From Oxford nanopore protocol - please refer to their protocol for further detail]
Prepare 5µg of high molecular weight genomic DNA (a range of 1-10µg can be used but we have found that higher DNA input works better).
?. [Preparing input DNA *From Oxford nanopore protocol - please refer to their protocol for further detail]
Prepare DNA in nuclease free water at a 24µl total volume in a PCR tube. Flick to resuspend and spin down.
?. [Preparing cas9 ribonucleoprotein complexes (RNPs) ]
Preheat thermal cycler to  . Thaw an aliquot of NEB CutSmart Buffer, mix by vortexing, and place on ice.
95 °C
?. [Preparing cas9 ribonucleoprotein complexes (RNPs) ]
In a PCR tube add in the following order (ensure crRNA & tracrRNA are equimolar - 100µM):Duplex Buffer8µLcrRNA pool (100µM)1µLtracrRNA (100µM)1µLPipette the mixture up and down. Place in thermal cycler for 5 mins at
Duplex Buffer8µLcrRNA pool (100µM)1µLtracrRNA (100µM)1µL
95 °C
?. [Preparing cas9 ribonucleoprotein complexes (RNPs) ]
Cool to room temperature and spin down to pool liquids - the crRNA : tracrRNA complexes will now be annealed.
?. [Forming RNPs]
In a 1.5ml Eppendorf tubeAnnealed tracrRNA : crRNA complex 10µL10x NEB cutsmart buffer10µLNuclease free H2O79.2µLPyrogenes Hifi Cas9 nuclease0.8µLTotal Volume 100µLMix by flicking the tube, and incubate at room temperature for 30 mins. Place on ice. The RNP complexes should now be formed.
Annealed tracrRNA : crRNA complex 10µL10x NEB cutsmart buffer10µLNuclease free H2O79.2µLPyrogenes Hifi Cas9 nuclease0.8µLTotal Volume 100µL
?. [Phosphorylate genomic DNA]
In a PCR tube add:10x CutSmart Buffer3µLYour Genomic DNA24µLTotal reaction Volume 27µLMix gently by flicking the tube and then spin down.
10x CutSmart Buffer3µLYour Genomic DNA24µLTotal reaction Volume 27µL
?. [Phosphorylate genomic DNA]
Add 3µl of CIP to the tubeUsing a Thermocycler, incubate at  for , for   then hold at  .
37 °C
80 °C
20 °C
?. [Cleave and dA-tail the target DNA]
Thaw dATP tube, vortex to mix, and place on ice.
?. [Cleave and dA-tail the target DNA]
In a 1.5ml Eppendorf lobind tube make up dATP mix to 10mM. Vortex to mix and spin down (e.g. 1µl of dATP to 9µl nuclease free water for a 100mM stock), then place on ice.
?. [Cleave and dA-tail the target DNA]
To the PCR tubes containing the 30µl of desphophorylated DNA add:Desphosphorylated genomic DNA sample30µlCas9 RNPs10µl10 mM dATP1µlNEB Taq polymerase1µlTotal reaction volume 42µlCarefully mix the contents by inversion of tube, spin down and place in thermal cycler.Incubate sample for the following cycle:  for  for   and hold at   .
Desphosphorylated genomic DNA sample30µlCas9 RNPs10µl10 mM dATP1µlNEB Taq polymerase1µlTotal reaction volume 42µl
37 °C
72 °C
4 °C
?. [Adaptor Ligation]
Thaw Ligation Buffer (LNB) at room temperature, mix via pipetting due to viscosity. Place on ice.Thaw the adaptor mix (AMX), mix by flicking tube and pulse spin. Return to ice.Place Ampure XP beads at room temperature
?. [Adaptor Ligation]
Using a wide-bore tip transfer contents from the previous step from the PCR tube into a 1.5ml lobind eppendorf tube.
?. [Adaptor Ligation]
In a seperate 1.5mL Eppendorf Tube assemble:Ligation Buffer (LNB)20µlNuclease free water3µlNEBNext Quick T4 DNA ligase10µlAdapter Mix (AMX)5µlTotal reaction volume 38µlMix by pipetting up and down to ensure the viscous LNB is incorporated
Ligation Buffer (LNB)20µlNuclease free water3µlNEBNext Quick T4 DNA ligase10µlAdapter Mix (AMX)5µlTotal reaction volume 38µl
?. [Adaptor Ligation]
Add 20µl of this adapter ligation mix to the cleaved and dA-tailed sample in the other 1.5ml eppendorf tube. Mix gently by flicking up and down. immediately after mixing, add the remainder of the adapter ligation mixture to the cleaved and dA-tailed sample, which will yield an 80µl ligation mix.Mix by flicking the tube and pulse spin to collect droplets.Incubate mixture for   at room temperature.
?. [Purify adapter ligated DNA - AMpure XP bead purification]
Resuspend the AMpure XP beads by vortexing.Thaw the short fragment buffer (SFB) and EB from SQK-LSK109.
?. [Purify adapter ligated DNA - AMpure XP bead purification]
Add 1x volume (80µl) of TE pH8.0 to the ligation mix and mix by gentle flicking of the tube. Then add 0.3x volume (48uL) of AMPure XP beads to the ligation sample. Gently invert the tube and then spin down. Incubate sample at room temperature for   - do not pipette or agitate sample.
?. [Purify adapter ligated DNA - AMpure XP bead purification]
Spin down the sample and pellet on a magnetic rack, then pipette off the supernatant.
?. [Purify adapter ligated DNA - AMpure XP bead purification]
Using 250µl of Short Fragment Buffer (SFB), resuspend the beads and flick to incorporate. Return tube to magnetic rack. Allow beads to pellet.Remove supernatant via pipette and discard.  Repeat this SFB wash step.
?. [Purify adapter ligated DNA - AMpure XP bead purification]
After removal of the supernatant, let the sample dry for .  Add 13uL of Elution Buffer (EB). Incubate for   at room temperature. Pellet beads using the magnetic rack and remove 12uL of eluate to a clean 1.5mL Eppendorf DNA LoBind Tube.This prepared library will be used for loading into the flow cell. Store on ice until ready to load.
?. [Priming and loading the flow cell ]
Thaw sequencing buffer (SQB), loading beads (LB), Flush tether (FLT) and one tube of flush buffer (FB). After thawing place on ice.
?. [Priming and loading the flow cell ]
Mix sequencing buffer (SQB) and flush buffer (FB) tubes by vortexing, spin down and return to ice.Mix the flush tether (FLT) by pipetting, then return to ice.
?. [Priming and loading the flow cell ]
Open the lid of the MinION sequencing device and slide the flow cell's priming port cover clockwise so that the priming port is visible.With a P1000µl pipette set to 200µl insert tip into the priming port and turn the wheel until the dial shows 220-230µl whereby a small volume of buffer will be seen in the tip.
?. [Priming and loading the flow cell ]
Prepare the flow cell priming mix: add 30µl of flush tether (FLT) directly to the tube of flush buffer (FB) and mix by pipetting up and down.
?. [Priming and loading the flow cell ]
Load 800µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait
?. [Priming and loading the flow cell ]
Thoroughly mix the contents of the Loading beads (LB) via pipetting.
?. [Priming and loading the flow cell ]
In a new 1.5ml lobind eppendorf tube prepare the library for loading:Sequencing Buffer (SQB)25µlLoading Beads (LB)13µlDNA library12µlTotal volume50µl
Sequencing Buffer (SQB)25µlLoading Beads (LB)13µlDNA library12µlTotal volume50µl
?. [Priming and loading the flow cell ]
Gently lift the sample port cover to make the sample port accessible.Load 200µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles.
?. [Priming and loading the flow cell ]
Mix the prepared library by gently pipetting up and down immediately prior to loading.
?. [Priming and loading the flow cell ]
Add 50µl of sample to the flow cell via the sample port in a slow drop wise fashion. Ensure each drop flows into the port before adding the next.
?. [Priming and loading the flow cell ]
Gently replace the sample port cover, close the priming port and replace the MinION lid.
?. [Starting the sequencing run ]
Follow the steps as outlined by Oxford Nanopore to start the sequencing run.