Mar 20, 2024

Public workspaceCas9-targeted Nanopore sequencing (CANS)

DOI
dx.doi.org/10.17504/protocols.io.261ge48mjv47/v1
  • 1Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia;
  • 2All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya Str. 42, 127550 Moscow, Russia
Pavel Merkulov
Open access
DOI: dx.doi.org/10.17504/protocols.io.261ge48mjv47/v1
Protocol Citation: Pavel Merkulov, Ilya Kirov 2024. Cas9-targeted Nanopore sequencing (CANS). protocols.io https://dx.doi.org/10.17504/protocols.io.261ge48mjv47/v1
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: June 09, 2021
Last Modified: March 20, 2024
Protocol Integer ID: 50606
Keywords: Cas9, Nanopore, CANS
Funders Acknowledgement:
Grant from the Russian Science Foundation (RSF)
Grant ID: 22-74-10055
Abstract
Here we provide a protocol for Cas9-targeted Nanopore sequencing.
We successfully applied this method for targeted sequencing and DNA methylation profiling of genes in cereal genomes, as well as for insertions of transposable elements (inherited and somatic) in Arabidopsis.
Materials
Materials:

  • PCR kit

  • PCR and gel extraction kit

  • T7 in vitro transcription kit

  • Total RNA and miRNA isolation kit

  • 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.

  • Quick Calf Intestinal Phosphatase (NEB cat #M0525)

  • 1.5 ml Eppendorf DNA LoBind tubes

  • 0.2 ml thin-walled PCR tubes

  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)

  • Taq polymerase (NEB Cat # M0273)

  • dATP solution (100 mM) (NEB Cat # N0440S)

  • LSK109 components

  • Ampure XP beads (Beckman Coulter A63881)

  • Flow Cell Wash Kit (EXP-WSH003 or EXP-WSH004)

Equipment:

  • Thermal cycler

  • P100 pipette and tips

  • P10 pipette and tips

  • P20 pipette and tips

  • Vortex mixer

  • Water bath

  • Ice bucket with wet ice
In vitro transcription of sgRNAs
In vitro transcription of sgRNAs
2d
Design a specific oligonucleotide for synthesizing a single guide RNA (sgRNA) template according to desired cut site in your target sequence (~20 nucleotides length and must be followed by a protospacer adjacent motif (PAM) sequence of NGG).
AB
Specific oligoGGATCCTAATACGACTCACTATAGG[target sequence]GTTTTAGAGCTAGAA.
CRISPR RAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
T7 FGGATCCTAATACGACTCACTATAG
T7 RAAAAAAGCACCGACTCGG

1d
Combine following components for sgRNA template synthesis:
AB
ComponentVolume, μL
Specific oligo, 1 μM2
CRISPR R, 1 μM2
T7 F, 100 μM2
T7 R, 100 μM2
dNTP mix, 10 mM of each2
10x buffer10
High fidelity polymerase1
Nuclease-free water79
Total100


10m
Set the reaction with following program:
  1. 95°C- 2 min
  2. 30 cycles:
  • 98°C - 30 sec
  • 60°C - 30 sec
  • 72°C - 30 sec
3. 72°C - 1 min


2h
Check the structure of synthesized templates with agarose gel electrophoresis (single band for best results, but note that T7-sequences can lead to dimers forming).

1h
Purify your sgRNA template with your system of choice. We use a column-based kit for gel extraction (in the case of dimers or non-specific products) and PCR purification (in the case of a single band).
30m
Combine following components for T7 in vitro transcription of your sgRNA:

AB
ComponentVolume, μL
5x buffer10
25x DTT2
rNPT mix, 25 mM of each2
sgRNA template (500 ng)X
T7 RNA (150U/μL)1 μL
Nuclease-free waterto get 50 µl total volume
Total50

10m
Incubate your reaction at Temperature37 °C for Duration02:00:00 . The incubation time can also be extended up to Duration16:00:00 (overnight) to obtain a higher sgRNA yield.

2h
Purify your sgRNA template with your system of choice. We use a kit for the isolation of total RNA and microRNA.
30m
Check the structure of synthesized sgRNA with agarose gel electrophoresis (the number of bands depends on the secondary sgRNA structure).
1h
Preparing the Cas9 ribonucleoprotein complexes (RNPs)
Preparing the Cas9 ribonucleoprotein complexes (RNPs)
40m
Combine equimolar amounts of sgRNAs for a targeted fragment in a single tube.
1m
Add water to get Amount11 µL

30s
Heat and cool each sgRNAs to obtain pure monomers: Temperature95 °C for Duration00:03:00 , then cool to TemperatureRoom temperature for Duration00:02:00
CITATION
Dang Y, Jia G, Choi J, Ma H, Anaya E, Ye C, Shankar P, Wu H (2015). Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency.. Genome biology.
LINK
https://doi.org/10.1186/s13059-015-0846-3

5m
Incubation
To form Cas9 RNPs, assemble the components in the table in a 1.5 ml Eppendorf DNA LoBind tube in the following order:
AB
ReagentVolume (per one cleavage reaction)
Cas9 5x buffer3
Cas91
gRNA (50ng/μl ~ 1pmol/ul) in 11 μl water11
Total 15
2m
Mix thoroughly by flicking the tube
30s
Mix
Form the RNPs by incubating the tube at TemperatureRoom temperature for Duration00:30:00 , then return the RNPs on ice until required (proceed to the 'Dephosphorylating genomic DNA' section during this time)

30m
Incubation
Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
32m
Transfer 1-10 μg (with 5 μg recommended) genomic DNA into 0.2 mL tubes.
CITATION
Boas Pucker. Plant DNA extraction and preparation for ONT sequencing. protocols.io.
LINK
https://protocols.io/view/plant-dna-extraction-and-preparation-for-ont-seque-bcvyiw7w
ml thin-walled PCR tubes
1m
Adjust to Amount24 µL with nuclease-free water

30s
Mix thoroughly by flicking the tube avoiding unwanted shearing
30s
Mix
Spin down briefly in a microfuge
30s
Mix the Quick calf intestinal alkaline phosphatase (CIP) in the tube by pipetting up and down. Ensure that it is at TemperatureRoom temperature before use

30s
Pipetting
Mix
Assemble the following components in a clean 0.2 ml thin-walled PCR tube:
AB
ReagentVolume
NEB CutSmart Buffer (10x)4 µl
HMW genomic DNA (at ≥ 210 ng/µl)*24-30 µl
Waterto get 34 µl total volume
Total34 µl
1m
Mix gently by flicking the tube, and spin down
30s
Mix
Add Amount6 µL of CIP to the tube

30s
Mix gently by flicking the tube, and spin down
30s
Mix
Using a thermal cycler, incubate at Temperature37 °C for Duration00:30:00 , Temperature80 °C for Duration00:02:00 then hold at TemperatureRoom temperature

32m
Incubation
Cleaving and dA-tailing target DNA
Cleaving and dA-tailing target DNA
35m
Thaw the dATP tube, vortex to mix thoroughly, and place on ice
2m
Dilute dATP to concentration Concentration10 millimolar (mM) . In a 0.2 ml thin-walled PCR tube, make a Concentration10 millimolar (mM) dATP solution by adding Amount1 µL of the Concentration100 millimolar (mM) dATP stock to Amount9 µL of nuclease-free water. Vortex to mix, then spin down

1m
Spin down and place the tube of Taq polymerase on ice
30s
To the PCR tube containing Amount40 µL dephosphorylated DNA sample, add:
AB
ReagentVolume
Dephosphorylated genomic DNA sample (Section 2)40 µl
Cas9 RNPs (Section 1)15 µl
10 mM dATP1.5 µl
Taq polymerase1 µl
Total57.5 µl


5m
Carefully mix the contents of the tube by gentle inversion, then spin down and place the tube in the thermal cycler
30s
Mix
Using the thermal cycler, incubate at Temperature37 °C for 15-60 (Duration00:15:00 are recommended) minutes, then Temperature72 °C for Duration00:10:00 and hold at Temperature4 °C or return to the tube to ice
25m
Incubation
Adapter ligation
Adapter ligation
25m
Assemble the following at room temperature in a separate 1.5 ml Eppendorf DNA LoBind Tube, adding Adapter Mix (AMX) last, before you are ready to begin the ligation:
AB
ReagentVolume
Ligation Buffer (LNB)25 µl
Nuclease-free water5 µl
NEBNext Quick T4 DNA Ligase12.5 µl
Adapter Mix (AMX)*5 µl
Total47.5 µl
* The Adapter Mix (AMX) must be added last and immediately before the ligation step

2m
Critical
Mix by pipetting the above ligation mix thoroughly. Ligation Buffer (LNB) is very viscous, so the adapter ligation mix needs to be well-mixed
30s
Pipetting
Mix
Add Amount20 µL of the adapter ligation mix to the cleaved and dA-tailed sample. Mix gently by flicking the tube. Do not centrifuge the sample at this stage. Immediately after mixing, add the remainder (Amount27.5 µL ) of the adapter ligation mix to the cleaved and dA-tailed sample, to yield a Amount105 µL ligation mix

1m
Mix gently by flicking the tube, and spin down
30s
Mix
Incubate the reaction for Duration00:20:00 at TemperatureRoom temperature
Note
A white precipitate may form upon the addition of the adapter ligation mix to the dA-tailed DNA. Adding the ligation mixture in two parts helps to reduce precipitation. However, the presence of a precipitate does not necessarily indicate failure of ligation of the sequencing adapter to target molecule ends.


20m
AMPure XP bead purification
AMPure XP bead purification
1h
Add 1 volume (Amount105 µL ) of TE (Ph8.0 ) to the ligation mix. Mix gently by flicking the tube

1m
Mix
Add 0.3x volume (Amount63 µL ) of AMPure XP Beads to the ligation sample. The volume of beads is calculated based on the volume after the addition of TE. Mix gently by inversion. If any sample ends up in the lid, spin down the tube very gently, keeping the beads suspended in a liquid

30s
Incubate the sample for Duration00:10:00 at TemperatureRoom temperature
Note
Do not agitate or pipette the sample to prevent long DNA fragments stick to the magnetic beads (it may decrease the elution)



10m
Incubation
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant
2m
Pipetting
Wash the beads by adding either Amount250 µL Long Fragment Buffer (LFB) or Amount250 µL Short Fragment Buffer (SFB), depending on the size of your target molecule. Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard

2m
Wash
Repeat the previous step Go togo to step #41
2m
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow drying for Duration00:00:30 , but do not dry the pellet to the point of cracking

30s
Pipetting
Critical
Remove the tube from the magnetic rack and resuspend the pellet in Amount13 µL Elution Buffer (EB). Incubate for Duration00:10:00 at TemperatureRoom temperature
Note
For fragments > 30 kb, we recommend increasing the elution time to Duration00:30:00

10m
Incubation
Pellet the beads on a magnet until the eluate is clear and colorless
1m
Remove and retain Amount12 µL of eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube

1m
Pipetting
Critical
Prime a MinION flow cell as specified in Nanopore protocols, and finally load the library drop-wise through the Sample port (a detailed description including video documentation can be found here: Priming and loading the SpotON flow cell)
20m
Analyze
Citations
Step 12
Dang Y, Jia G, Choi J, Ma H, Anaya E, Ye C, Shankar P, Wu H. Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency.
https://doi.org/10.1186/s13059-015-0846-3
Step 16
Boas Pucker. Plant DNA extraction and preparation for ONT sequencing
https://protocols.io/view/plant-dna-extraction-and-preparation-for-ont-seque-bcvyiw7w