TITLE: SMRT-OTS
AUTHORS: Ida Hoijer, Josefin Johansson, Sanna Gudmundsson, Chen-Shan Chin, Ignas Bunikis, Susana Häggqvist, Anastasia Emmanouilidou, Maria Wilbe, Marcel den Hoed, Marie-Louise Bondeson, Lars Feuk, Ulf Gyllensten, Adam Ameur

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a protocol that detects Cas9 off-target sites </span><span style = "font-style:italic;">in vitro </span><span>using</span><span style = "font-style:italic;"> </span><span>SMRT sequencing</span><span style = "vertical-align:super;">1</span><span>. The protocol is a modified version described by Tsai et al (2017). Guide RNAs have been designed using CHOPCHOP (</span><a href="https://chopchop.cbu.uib.no" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://chopchop.cbu.uib.no/</span></a><span>).</span></div><div class = "text-block">1. Höijer, I., et al. (2020). "Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity." bioRxiv: 2020.2002.2009.940486.</div><div class = "text-block">2. Tsai, Y-C., et al. (2017). "Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions". bioRxiv: 203919.</div></div>

[STEPS]
?. [Shearing of genomic DNA]
Shear 10-20 µg of HMW genomic DNA to 8 kb using Megaruptor 2.
?. [Shearing of genomic DNA]
Add 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.
?. [Shearing of genomic DNA]
Place the tube on a magnetic rack until the beads collect on the side of the tube and the solution appears to be clear. Slowly pipette off the supernatant and discard. Be careful not to disturb the beads.
?. [Shearing of genomic DNA]
Wash the beads using 70% freshly prepared ethanol.     - Fill the tube with the ethanol    - Do not disurb the bead pellet    - Wait 30 seconds and pipette off the ethanol and discard
?. [Shearing of genomic DNA]
Repeat step 2.2
?. [Shearing of genomic DNA]
Remove any residual ethanol.    - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining ethanol using a P10 pipette
?. [Shearing of genomic DNA]
Add 40 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the sample.
?. [Shearing of genomic DNA]
Place the tube on the magnetic rack until the beads collect on the side of the tube and the solution appears clear. Slowly pipette off the supernatant and transfer to a new eppendorf tube.
?. [Quality control of fragmented DNA]
Measure concentration of the sample using Qubit and fragment size using Bioanalyzer or Tape station.
?. [SMRT bell library construction]
The following protocol is optimized for 5 µg of sheared and concentrated DNA. Scale the reaction if needed.
?. [SMRT bell library construction]
ExoVII treatmentAdd the following compontents to a new 1.5 ml eppendorf tube:        38 µl sheared DNA        5 µl DNA damage repair buffer        0.5 µl NAD+         5 µl ATP high        0.5 µl dNTP        1 µl ExoVIIMix by gently flicking the tube, spin down. Incubate at 37ºC for 15 min. Place the tube on ice.
?. [SMRT bell library construction]
DNA damage repairAdd the following component to the EvoVII treated sample:        2 µl DNA damage repair mixMix by flicking the tube gently, spin down. Incubate at 37ºC for 20 min. Place the tube on ice for 1-5 min.
?. [SMRT bell library construction]
End repairAdd the following component to the DNA damaged repaired treated sample:        2.5 µl End repair mixMix by gently flicking the tube, spin down. Incubate at 25ºC for 5 min. Place the tube on ice.
?. [SMRT bell library construction]
Purify the DNA with AMPure PB beadsAdd 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.
?. [SMRT bell library construction]
Place the tube on a magnetic rack until the beads collect on the side of the tube and the solution appears to be clear. Slowly pipette off the supernatant and discard. Be careful not to disturb the beads.
?. [SMRT bell library construction]
Wash the beads using 70% freshly prepared ethanol.     - Fill the tube with the ethanol    - Do not disurb the bead pellet    - Wait 30 seconds and pipette off the ethanol and discard
?. [SMRT bell library construction]
Repeat step 8.2
?. [SMRT bell library construction]
Remove any residual ethanol.     - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining ethanol using a P10 pipette
?. [SMRT bell library construction]
Add 32 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the sample.
?. [SMRT bell library construction]
Place the tube on the magnetic rack until the beads collect on the side of the tube and the solution appears clear. Slowly pipette off the supernatant and transfer to a new eppendorf tube.
?. [SMRT bell library construction]
Adapter ligationAdd the following reagents to the end repaired and purfied DNA sample:        1 µl Blunt adapterMix by the tube by gently flicking the tube, spin down.        4 µl Template prep buffer        2 µl ATP lowMix by the tube by gently flicking the tube, spin down.        1 µl LigaseMix by gently flicking the tube, spin down. Incubate at 25ºC overnight. Incubate at 65ºC for 10 min to inactivate the ligase. Place the tube on ice.
?. [SMRT bell library construction]
Exonuclease III and VII treatmentAdd the following compontents to the adapter ligated sample:        1 µl ExoIII        1 µl ExoVIIMix by gently flicking the tube, spin down. Incubate at 37ºC for 1 hour, then place the tube on ice. It is important to immediately proceed to purification after this step.
?. [SMRT bell library construction]
Purify the DNA with AMPure PB beadsAdd 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.
?. [SMRT bell library construction]
Place the tube on a magnetic rack until the beads collect on the side of the tube and the solution appears to be clear. Slowly pipette off the supernatant and discard. Be careful not to disturb the beads.
?. [SMRT bell library construction]
Wash the beads using 70% freshly prepared ethanol.     - Fill the tube with the ethanol    - Do not disurb the bead pellet    - Wait 30 seconds and pipette off the ethanol and discard
?. [SMRT bell library construction]
Repeat step 11.2
?. [SMRT bell library construction]
Remove any residual ethanol.     - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining ethanol using a P10 pipette
?. [SMRT bell library construction]
Add 40 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the sample.
?. [SMRT bell library construction]
Place the tube on the magnetic rack until the beads collect on the side of the tube and the solution appears clear. Slowly pipette off the supernatant and transfer to a new 0.2 ml PCR tube.
?. [Quality control of SMRT bell library]
Measure concentration of the sample using Qubit and fragment size using Bioanalyzer or Tape station.
?. [Size selction of SMRT bell library]
Perform size selection of the SMRT bell library using a 1.5-4 kb cut-off (depending on how the fragment profile of the SMRT bell library) using the "0.75% DF marker S1 high pass 6-10 kb vs3" casette.
?. [SMRT bell library construction]
Lambda exonuclease and exonuclease I treatmentAdd the following components to the Exo III and VII treated sample:        5 µl Exonuclease I reaction buffer (10X)        4 µl Lambda exonuclease (5U/µl)        1 µl Exonuclease I (20U/µl)Mix by gently flicking the tube, spin down. Incubate at 37ºC for 1 hour, 75ºC for 10 min and then hold at 4ºC using a thermocycler. Carefully tranfer the sample to a new 1.5 ml eppendorf tube.
?. [SMRT bell library construction]
Purify the DNA with AMPure PB beadsAdd 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.
?. [SMRT bell library construction]
Place the tube on a magnetic rack until the beads collect on the side of the tube and the solution appears to be clear
?. [SMRT bell library construction]
Slowly pipette off the supernatant and discard. Be careful not to disturb the beads.
?. [SMRT bell library construction]
Wash the beads using 70% freshly prepared ethanol.     - Fill the tube with the ethanol    - Do not disurb the bead pellet    - Wait 30 seconds and pipette off the ethanol and discard
?. [SMRT bell library construction]
Repeat step 13.3
?. [SMRT bell library construction]
Remove any residual ethanol.     - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining ethanol using a P10 pipette
?. [SMRT bell library construction]
Add 32 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the sample
?. [SMRT bell library construction]
Place the tube on the magnetic rack until the beads collect on the side of the tube and the solution appears clear. Slowly pipette off the supernatant and transfer to a new 1.5 ml eppendorf tube.
?. [Size selction of SMRT bell library]
Purify the DNA with AMPure PB beadsAdd 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.
?. [Size selction of SMRT bell library]
Place the tube on a magnetic rack until the beads collect on the side of the tube and the solution appears to be clear
?. [Size selction of SMRT bell library]
Slowly pipette off the supernatant and discard. Be careful not to disturb the beads.
?. [Size selction of SMRT bell library]
Wash the beads using 70% freshly prepared ethanol.     - Fill the tube with the ethanol    - Do not disurb the bead pellet    - Wait 30 seconds and pipette off the ethanol and discard
?. [Size selction of SMRT bell library]
Repeat step 16.3
?. [Size selction of SMRT bell library]
Remove any residual ethanol.     - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining ethanol using a P10 pipette
?. [Size selction of SMRT bell library]
Add 22 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the sample
?. [Size selction of SMRT bell library]
Place the tube on the magnetic rack until the beads collect on the side of the tube and the solution appears clear. Slowly pipette off the supernatant and transfer to a new 1.5 ml eppendorf tube.
?. [Quality control of size selected SMRT bell library]
Measure concentration of the sample using Qubit and fragment size using Bioanalyzer or Tape station.
?. [Preparation of guide RNAs]
Resuspend crRNA to 10 nM in nuclease-free water and tracrRNA to 10 nM in tracr buffer.
?. [Preparation of guide RNAs]
Guide RNA preparationAdd the following components in a new 0.2 ml PCR tube:        5 µl crRNA (10 nM)        5 µl tracrRNA (10 nM)Incubate at 50ºC for 5 min, ramp down to 25ºC at 0.1ºC/s, 25ºC for 5 min and hold at 4ºC.
?. [Preparation of guide RNAs]
For a multiplexed approach; combine equal volumes of gRNAs in a new eppendorf tube. Do not keep left over gRNA for future experiments.
?. [Cas9 digestion]
Reconstitute 5X CRISPR buffer by combining 95 µl 5X CRISPR buffer with 5 µl Reducing agent in a new 1.5 ml eppendorf tube. Do not keep the remaining buffer for future experiments.
?. [Cas9 digestion]
Add the following compontents to a 0.2 ml PCR tube:        10 µl reconstituted 5X CRISPR buffer        30-X µl nuclease-free water        2 µl guide RNA        0.5 µl Cas9 nucleaseMix by gently flicking the tube, spin down. Incubate at 37ºC for 10 min. Immediately place the tube on ice.
?. [Cas9 digestion]
1 µg of size selected SMRT bell library is needed in this step. Calculate the volume (X) for 1 µg of the sample. In step 23, the volume of nuclease-free water is 30-X µl.
?. [Cas9 digestion]
Add the following component to the same tube:        7.5 µl heparin (20 µg/µl)Mix gently by flicking the tube, spin down. Incubate at 37ºC for 3 min. Immediately place the tube on ice.
?. [Cas9 digestion]
Add 1 µg (X µl) of size selected SMRT bell library to the PCR tube. Mix gently by flicking the tube, spin down. Incubate at 37ºC for 1 hour. Immediately place the tube on ice.
?. [Cas9 digestion]
Add 10 µl of 0.5 M EDTA to stop the reaction. Mix gently by flicking the tube, spin down. Place the tube on ice.
?. [AMPure PB purification of Cas9 digested sample]
Add 0.5x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.
?. [Cas9 digestion]
Carefully transfer the sample to a new 0.5 ml eppendorf tube.
?. [AMPure PB purification of Cas9 digested sample]
Place the tube on a magnetic rack and allow the beads to collect for at least 5 min or until the solution appears clear. Slowly pipette off the supernatant and discard. Be careful not to disturb the beads.
?. [AMPure PB purification of Cas9 digested sample]
Wash the beads using 80% freshly prepared ethanol.     - Fill the tube with the ethanol    - Do not disurb the bead pellet    - Wait 30 seconds and pipette off the ethanol and discard
?. [AMPure PB purification of Cas9 digested sample]
Repeat step 30 twice.
?. [AMPure PB purification of Cas9 digested sample]
Remove any residual ethanol.    - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining ethanol using a P10 pipette
?. [AMPure PB purification of Cas9 digested sample]
Add 39.5 µl Elution buffer to the beads. Mix until the solution is homogenous by flicking the tube. Incubate at room temperature for 5 min. Incubate at 2000 rpm for 2 min using a vortex mixer to elute the sample.
?. [AMPure PB purification of Cas9 digested sample]
Place the tube on the magnetic rack until the beads collect on the side of the tube and the solution appears clear. Slowly pipette off the supernatant and transfer to a new 0.5 ml eppendorf tube.
?. [Quality control of the Cas9 digested sample]
Use 1 µl of sample to measure DNA concentration using Qubit dsDNA HS assay kit. The typical DNA yield is between approximately 80-90% of the input amount into the Cas9 digestion.
?. [Capture adapter ligation]
Add the following reagents to the Cas9 digested DNA sample:        1 µl Capture adapter (annealed)Mix by the tube by gently flicking the tube, spin down.        5 µl T4 DNA ligase reaction buffer (10x)Mix by the tube by gently flicking the tube, spin down.        1.5 µl T4 DNA ligaseMix gently by flicking the tube, spin down. Incubate at 16ºC overnight.If no 0.5 ml heat block or thermocycler is available, place a 1.5 ml eppendorf tube in the heat block and add 1 ml nuclease-free water to the tube. Place the 0.5 ml tube with the ligation reaction in the water-filled tube. The same applies to step 37, 39 and 85.
?. [Capture adapter ligation]
Incubate at 65ºC for 10 min to inactivate the ligase.
?. [Capture adapter ligation]
Centrifuge the sample at 14 000 rcf for 5 min in a micro centrifuge. Transfer the supernatant to a new 0.5 ml eppendorf tube. Proceed to the next section.
?. [Exonuclease treatment]
Add the following components to the capture adapter ligated DNA sample:        1 µl ExoIII        1 µl ExoVIIIncubate at 37ºC for 1 hour, the place the tube on ice. Proceed immediately to the next step.
?. [2x AMPure PB purification of exonuclease treated sample]
Perform two consecutive AMPure PB bead purifications. Elute in 50 µl Elution buffer after the first purification and 21 µl Elution buffer after the second purification.
?. [2x AMPure PB purification of exonuclease treated sample]
Add 0.5x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.
?. [2x AMPure PB purification of exonuclease treated sample]
Place the tube on a magnetic rack and allow the beads to collect for at least 5 min or until the solution appears clear. Slowly pipette off the supernatant and discard. Be careful not to disturb the beads.
?. [2x AMPure PB purification of exonuclease treated sample]
Wash the beads using 80% freshly prepared ethanol.     - Fill the tube with the ethanol    - Do not disurb the bead pellet    - Wait 30 seconds and pipette off the ethanol and discard
?. [2x AMPure PB purification of exonuclease treated sample]
Repeat step 43 twice.
?. [2x AMPure PB purification of exonuclease treated sample]
Remove any residual ethanol.    - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining ethanol using a P10 pipette
?. [2x AMPure PB purification of exonuclease treated sample]
Add 50/22 µl Elution buffer to the beads. Mix until the solution is homogenous by flicking the tube. Incubate at room temperature for 5 min. Incubate at 2000 rpm for 2 min using a vortex mixer to elute the sample.
?. [2x AMPure PB purification of exonuclease treated sample]
Place the tube on the magnetic rack until the beads collect on the side of the tube and the solution appears clear. Slowly pipette off the supernatant and transfer to a new 0.5 ml eppendorf tube.The purified DNA can be stored at 4ºC overnight or at -20ºC for longer duration before proceeding to the next step.
?. [Quality control of exonuclease treated sample]
Use 1 µl of DNA sample to measure the DNA concentration using the Qubit dsDNA HS assay kit. Typical DNA yield is approximately 50-70% of the input amount going into Cas9 digestion.
?. [Enrichment of assymmetric SMRTbell templates]
Tips for success:    - Bring MagBeads to room temperature before use.    - Mix the MagBeads well, until homogeneous, before dispensing. Pipette the bead solution slowly as the bead mixture is viscous. Precise volume are critical to the purification process.    - Minimize bubble formation during the mixing of the beads and buffer by gently tapping the finger on the tube and/or magnet pulling.    - Do not pellet the beads when spinning to collect liquid.    - Bring the buffers to room temperature before use in the MagBead washing step (Step 60-67).
?. [MagBead preparation]
Add 50 µl of MagBeads to a 1.5 ml eppendorf tube.
?. [MagBead preparation]
Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.
?. [MagBead preparation]
Add 50 µl of MagBead Wash Buffer v2 to the tube and mix. Spin briefly.
?. [MagBead preparation]
Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.
?. [MagBead preparation]
Add 50 µl of MagBead Binding Buffer v2 to the tube and mix. Spin briefly.
?. [MagBead preparation]
Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.
?. [MagBead preparation]
Add 7 µl of MagBead Wash Buffer v2 to the tube and mix. Spin briefly. Proceed to the next step.
?. [MagBead binding]
Add 21 µl of the CRISPR-Cas9 generated SMRTbell library to 7 µl of the prepared MagBeads. Gently mix, ensure the beads are completely resuspended. Spin briefly.
?. [MagBead binding]
Incubate at 4ºC for 2 hours with gentle rotation in a tube rotator.
?. [MagBead binding]
Spin briefly and place the tube on ice. Proceed to the next step.
?. [MagBead washing]
Note: If multiple samples, process one sample at a time starting at Step 60. Keep addtional samples on ice until ready to process.Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.
?. [MagBead washing]
Add 100 µl of room temperature MagBead bindning buffer v2 to the tube. Mix gently and spin briefly.
?. [MagBead washing]
Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.
?. [MagBead washing]
Add 100 µl of room temperature MagBead bindning buffer v2 to the tube. Mix gently and spin briefly. Transfer the soltion to a new 1.5 ml eppendorf tube.
?. [MagBead washing]
Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.
?. [MagBead washing]
Add 100 µl of room temperature MagBead bindning buffer v2 to the tube. Mix gently and spin briefly.
?. [MagBead washing]
Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.
?. [MagBead washing]
Add 7 µl Elution buffer to the tube to elute the captured SMRTbell library. Mix gently and spin briefly.Note: Keep samples in Elution buffer at room temperature if processing additional samples.
?. [Sample elution]
Note: If multiple samples, process one sample at a time starting at Step 69. Keep additional samples at 50ºC.Incubate the sample at 50ºC for 10 min in an Eppendorf ThermoMixer at full mixing speed.
?. [Sample elution]
Spin briefly to collect the liquid.
?. [Sample elution]
Collect the beads at the side of the tube in a magnetic rack. Quickly transfer the supernatant to a new tube before the solution cools down. Place the tube on ice.
?. [Sample elution]
Samples can be stored overnight at 4ºC or at -20ºC for longer duration before proceeding to the next step.
?. [Primer annealing]
Prepare diluted Sequencing primer v4 before reaction setup by adding 1 µl primer to 29 µl nuclease-free water.
?. [Primer annealing]
Add the following components to a 0.2 ml PCR tube:    18 µl 10X Primer buffer v2    9 µl diluted Sequencing primer v4Mix gently by flicking the tube. Spin briefly. Incubate at 80ºC for 2 minutes and hold at 4ºC using a thermocycler.
?. [Primer annealing]
Add the follwing to a new 0.2 ml PCR tube:    2.7 µl conditioned sequencing primer    6.3 µl enriched SMRTbell libraryIncubate at room temperatrue for 1 hr. Place the tube on ice.
?. [Polymerase binding]
Prepare diluted Sequel polymerase 3.0 just prior to use by adding 1 µl polymerase to 29 µl Sequel binding buffer v1 and keep on ice. Diluted polymerase must be used immediately. Discard the unused portion.
?. [Polymerase binding]
Add the following components to the tube containing 9 µl primer annealed SMRTbell library:    1.5 µl Sequel binding buffer v1    1.5 µl DTT    1.5 µl dNTP v3Mix before proceeding    1.5 µl Diluted Sequel polymerase 3.0Mix gently by flicking the tube. Spin down. Incubate at 30ºC for 4 hours and hold at 4ºC using a thermocycler.
?. [Sequel sequencing preparation]
Make two 100-fold serial dilutions of the Sequel DNA internal control 3.0 in MagBead binding buffer v2:    - Add 1 µl control complex to 99 µl MagBead binding buffer v2.    - Mix well by flicking the tube. Spin down.    - Add 1 µl of the diluted control complex to 99 µl MagBead binding buffer v2.    - Mix well by flicking the tube. Spin down.
?. [Sequel sequencing preparation]
Dispense 2.8 µl of the final control complex dilution to a sequencing sample plate for each sample. Cover the sample plate and keep on ice until ready to add the purified sample complex.
?. [Sample complex purification]
Carefully transfer the 15 µl of samplex complex to a new 0.5 ml eppendorf tube.
?. [Sample complex purification]
Add 35 µl MagBead binding buffer v2 to the tube containing 15 µl of sample complex.
?. [Sample complex purification]
Add 30 µl AMPure PB beads to the tube containing 50 µl diluted sample complex. Mix well by flicking the tube. Spin down.Incubate at room temperature for 5 min.
?. [Sample complex purification]
Place the tube on a magnetic rack for 2 min and slowly remove the supernatant and discard.
?. [Sample complex purification]
Immediately resuspend the beads in 41.1 µl room temperature MagBead binding buffer v2. Mix well by flicking the tube. Spin down.Incubate in room temperature for 15 min.
?. [Sample complex purification]
Place the tube on a magnetic rack for 1 min and slowly remove the supernatant and transfer to the well in the sequencing sample plate containing the control complex.
?. [Sample complex purification]
Add an additional 41.1 µl room temperature MagBead binding buffer v2 to the tube containing the beads. Mix well by flicking the tube. Spin down.Incubate at 37ºC for 15 min (same technique described in Step 36 can be used).
?. [Sample complex purification]
Place the tube on a magnetic rack for 1 min and slowly remove the supernatant and transfer to the well in the sequencing sample plate containing the control complex and the first sample eluate.
?. [Sample complex purification]
Cover the sample plate at keep on ice until ready to sequence.
?. [Sample complex purification]
Sequence with diffusion loading, 4 hr immobilization time and 10 hr movie time.
?. [Data analysis]
Because of the assymmetric nature of the SMRTbells recalling of adapters has to be performed, using the following tool:https://github.com/PacificBiosciences/recalladapters
?. [Data analysis]
Perform CCS analysis on the recalled data.
?. [Data analysis]
Align the CCS reads to a reference using minimap2.
?. [Data analysis]
Use the mapped reads for detection of off-target sites using our custom tool Insider:https://github.com/UppsalaGenomeCenter/InSiDeR