SMRT-OTS

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

Sep 23, 2020

SMRT-OTS

This protocol is a draft, published without a DOI.

Ida Hoijer¹,
Josefin Johansson²,
Sanna Gudmundsson³,
Chen-Shan Chin⁴,
Ignas Bunikis²,
Susana Häggqvist²,
Anastasia Emmanouilidou⁵,
Maria Wilbe⁵,
Marcel den Hoed⁵,
Marie-Louise Bondeson⁵,
Lars Feuk⁵,
Ulf Gyllensten⁵,
Adam Ameur⁵

¹SciLifeLab, Uppsala University;
²Uppsala University;
³Broad Institute of Massachusetts Institute of Technology and Harvard;
⁴Foundation for Biological Science;
⁵Uppsala Universtiy

Ida Hoijer

SciLifeLab, Uppsala University

Protocol Citation: 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 2020. SMRT-OTS. protocols.io https://protocols.io/view/smrt-ots-bjugkntw

Manuscript citation:

https://www.biorxiv.org/content/10.1101/2020.02.09.940486v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: August 17, 2020

Last Modified: September 23, 2020

Protocol Integer ID: 40552

Abstract

This is a protocol that detects Cas9 off-target sites in vitro using SMRT sequencing1. The protocol is a modified version described by Tsai et al (2017). Guide RNAs have been designed using CHOPCHOP (https://chopchop.cbu.uib.no/).

1. Höijer, I., et al. (2020). "Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity." bioRxiv: 2020.2002.2009.940486.

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.

Materials

MATERIALS
ReagentT4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S 
ReagentLambda Exonuclease - 1,000 unitsNew England BiolabsCatalog #M0262S 
ReagentCas9 Nuclease, S. pyogenes - 400 pmolNew England BiolabsCatalog #M0386T 
ReagentUltraPure 0.5M EDTA, pH 8.0Thermo Fisher ScientificCatalog #15575-038 
ReagentExonuclease III (E. coli)New England BiolabsCatalog #M0206 
ReagentExonuclease VIINew England BiolabsCatalog #M0379 
ReagentAlt-R® CRISPR-Cas9 tracrRNAIDT TechnologiesCatalog #1072532 
ReagentAlt-R® CRISPR-Cas9 crRNAIDT Technologies 
Reagent0.75% Agarose High Pass DNA Size Selection Gel casettessage scienceCatalog #PAC-20KB 
ReagentT4 DNA ligase HCThermo Fisher ScientificCatalog #EL0013 
ReagentMagBead Kit v2Pacific BiosciencesCatalog #100-676-500 
ReagentHeparin 20 µg/µl 
ReagentSMRTbell Template Prep Kit 1.0-SPv3Pacific BiosciencesCatalog #100-991-900 
ReagentAmpure PB 5 mLsPacific BiosciencesCatalog #100-265-900 
ReagentExonuclease INew England BiolabsCatalog #M0293S 
ReagentSequencing Primer v4Pacific Biosciences 
Capture adapter: 
    Phosp-ATC TCT CTC TTA AAA AAA AAA AAA AAA AAA AAA ATT GAG AGA GAT

Annealing of Capture adapter

Prepare 10x annealing buffer:
    100 mM Tris-HCl, ph 7.5
    1 M NaCl

Resuspend the adapter to 20 µM in 1x annealing buffer. Anneal as follows:
Incubate at 80°C for 2 min, then ramp temperature down to 25°C at a rate of 0.1°C/sec. Hold at 4°C.
Store the annealed adapter at -20°C.

tracr buffer:
    40 mM HEPES, pH 7.5
    40 mM KCl
    10% Glycerol

Reducing agent:
    100 mM TCEP in water

5X CRISPR buffer:
    100 mM HEPES, pH 7.5
    500 mM KCl
    25% Glycerol
    25 mM MgCl2

 

 

Shearing of genomic DNA

Shear 10-20 µg of HMW genomic DNA to 8 kb using Megaruptor 2. 

Add 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.

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.

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

Repeat step 2.2

Remove any residual ethanol.

    - Spin the tube briefly and put back on the magnetic rack
    - Pipette off any remaining ethanol using a P10 pipette

Add 40 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the 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 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.

ExoVII treatment

Add 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 ExoVII

Mix by gently flicking the tube, spin down. Incubate at 37ºC for 15 min. Place the tube on ice.

DNA damage repair

Add the following component to the EvoVII treated sample:

        2 µl DNA damage repair mix

Mix by flicking the tube gently, spin down. Incubate at 37ºC for 20 min. Place the tube on ice for 1-5 min.

End repair

Add the following component to the DNA damaged repaired treated sample:

        2.5 µl End repair mix

Mix by gently flicking the tube, spin down. Incubate at 25ºC for 5 min. Place the tube on ice.

Purify the DNA with AMPure PB beads

Add 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.

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.

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

Repeat step 8.2

Remove any residual ethanol. 


    - Spin the tube briefly and put back on the magnetic rack
    - Pipette off any remaining ethanol using a P10 pipette

Add 32 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the 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 eppendorf tube.

Adapter ligation

Add the following reagents to the end repaired and purfied DNA sample:

        1 µl Blunt adapter
Mix by the tube by gently flicking the tube, spin down.
        4 µl Template prep buffer
        2 µl ATP low
Mix by the tube by gently flicking the tube, spin down.
        1 µl Ligase

Mix 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.

Exonuclease III and VII treatment

Add the following compontents to the adapter ligated sample:

        1 µl ExoIII
        1 µl ExoVII

Mix 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.

Purify the DNA with AMPure PB beads

Add 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.

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.

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

Repeat step 11.2

Remove any residual ethanol. 

    - Spin the tube briefly and put back on the magnetic rack
    - Pipette off any remaining ethanol using a P10 pipette

Add 40 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the 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 0.2 ml PCR tube.

Lambda exonuclease and exonuclease I treatment

Add 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.

Purify the DNA with AMPure PB beads

Add 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.

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.

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

Repeat step 13.3

Remove any residual ethanol. 

    - Spin the tube briefly and put back on the magnetic rack
    - Pipette off any remaining ethanol using a P10 pipette

Add 32 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the 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 1.5 ml eppendorf 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.

Purify the DNA with AMPure PB beads

Add 0.45x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.

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.

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

Repeat step 16.3

Remove any residual ethanol. 

    - Spin the tube briefly and put back on the magnetic rack
    - Pipette off any remaining ethanol using a P10 pipette

Add 22 µl Elution buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the 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 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.

Guide RNA preparation

Add 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.

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.

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.

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 nuclease

Mix by gently flicking the tube, spin down. Incubate at 37ºC for 10 min. Immediately place the tube on ice.

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.

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.

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.

Carefully transfer the sample to a new 0.5 ml eppendorf tube.

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.

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.

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

Repeat step 30 twice.

Remove any residual ethanol.


    - Spin the tube briefly and put back on the magnetic rack
    - Pipette off any remaining ethanol using a P10 pipette

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.

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 ligase

Mix 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.

Incubate at 65ºC for 10 min to inactivate the ligase.

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 ExoVII

Incubate 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.

Add 0.5x AMPure PB beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.

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.

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

Repeat step 43 twice.

Remove any residual ethanol.


    - Spin the tube briefly and put back on the magnetic rack
    - Pipette off any remaining ethanol using a P10 pipette

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.

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.

Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.

Add 50 µl of MagBead Wash Buffer v2 to the tube and mix. Spin briefly.

Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.

Add 50 µl of MagBead Binding Buffer v2 to the tube and mix. Spin briefly.

Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard.

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.

Incubate at 4ºC for 2 hours with gentle rotation in a tube rotator.

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. 

Add 100 µl of room temperature MagBead bindning buffer v2 to the tube. Mix gently and spin briefly.

Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard. 

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.

Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard. 

Add 100 µl of room temperature MagBead bindning buffer v2 to the tube. Mix gently and spin briefly.

Collect the beads to the side of the tube in a magnetic rack. Remove the supernatant and discard. 

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.

Spin briefly to collect the liquid.

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.

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.

Add the following components to a 0.2 ml PCR tube:

    18 µl 10X Primer buffer v2
    9 µl diluted Sequencing primer v4

Mix gently by flicking the tube. Spin briefly. Incubate at 80ºC for 2 minutes and hold at 4ºC using a thermocycler.  

Add the follwing to a new 0.2 ml PCR tube:

    2.7 µl conditioned sequencing primer
    6.3 µl enriched SMRTbell library

Incubate 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.

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 v3
Mix before proceeding
    1.5 µl Diluted Sequel polymerase 3.0

Mix 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.

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.

Add 35 µl MagBead binding buffer v2 to the tube containing 15 µl of sample complex.

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.

Place the tube on a magnetic rack for 2 min and slowly remove the supernatant and discard.

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.

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.

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).

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.

Cover the sample plate at keep on ice until ready to sequence.

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

Perform CCS analysis on the recalled data.

Align the CCS reads to a reference using minimap2.

Use the mapped reads for detection of off-target sites using our custom tool Insider:

https://github.com/UppsalaGenomeCenter/InSiDeR

Public workspaceSMRT-OTS

SMRT-OTS