Mar 20, 2024
Cas9-targeted Nanopore sequencing (CANS)
- Pavel Merkulov1,2,
- Ilya Kirov1,2
- 1Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia;
- 2All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya Str. 42, 127550 Moscow, Russia
- High molecular weight DNA extraction from all kingdoms
- Long Read Club
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 Acknowledgements:
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
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).
| A | B | |
| Specific oligo | GGATCCTAATACGACTCACTATAGG[target sequence]GTTTTAGAGCTAGAA. | |
| CRISPR R | AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC | |
| T7 F | GGATCCTAATACGACTCACTATAG | |
| T7 R | AAAAAAGCACCGACTCGG |
1d
Combine following components for sgRNA template synthesis:
| A | B | |
| Component | Volume, μL | |
| Specific oligo, 1 μM | 2 | |
| CRISPR R, 1 μM | 2 | |
| T7 F, 100 μM | 2 | |
| T7 R, 100 μM | 2 | |
| dNTP mix, 10 mM of each | 2 | |
| 10x buffer | 10 | |
| High fidelity polymerase | 1 | |
| Nuclease-free water | 79 | |
| Total | 100 |
10m
Set the reaction with following program:
- 95°C- 2 min
- 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:
| A | B | |
| Component | Volume, μL | |
| 5x buffer | 10 | |
| 25x DTT | 2 | |
| rNPT mix, 25 mM of each | 2 | |
| sgRNA template (500 ng) | X | |
| T7 RNA (150U/μL) | 1 μL | |
| Nuclease-free water | to get 50 µl total volume | |
| Total | 50 |
10m
Incubate your reaction at 37 °C for 02:00:00 . The incubation time can also be extended up to 16:00:00 (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
40m
Combine equimolar amounts of sgRNAs for a targeted fragment in a single tube.
1m
Add water to get 11 µL
30s
Heat and cool each sgRNAs to obtain pure monomers: 95 °C for 00:03:00 , then cool to Room temperature for 00:02:00
CITATION
5m
To form Cas9 RNPs, assemble the components in the table in a 1.5 ml Eppendorf DNA LoBind tube in the following order:
| A | B | |
| Reagent | Volume (per one cleavage reaction) | |
| Cas9 5x buffer | 3 | |
| Cas9 | 1 | |
| gRNA (50ng/μl ~ 1pmol/ul) in 11 μl water | 11 | |
| Total | 15 |
2m
Mix thoroughly by flicking the tube
30s
Form the RNPs by incubating the tube at Room temperature for 00:30:00 , then return the RNPs on ice until required (proceed to the 'Dephosphorylating genomic DNA' section during this time)
30m
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
32m
Transfer 1-10 μg (with 5 μg recommended) genomic DNA into 0.2 mL tubes.
CITATION
ml thin-walled PCR tubes
1m
Adjust to 24 µL with nuclease-free water
30s
Mix thoroughly by flicking the tube avoiding unwanted shearing
30s
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 Room temperature before use
30s
Assemble the following components in a clean 0.2 ml thin-walled PCR tube:
| A | B | |
| Reagent | Volume | |
| NEB CutSmart Buffer (10x) | 4 µl | |
| HMW genomic DNA (at ≥ 210 ng/µl)* | 24-30 µl | |
| Water | to get 34 µl total volume | |
| Total | 34 µl |
1m
Mix gently by flicking the tube, and spin down
30s
Add 6 µL of CIP to the tube
30s
Mix gently by flicking the tube, and spin down
30s
Using a thermal cycler, incubate at 37 °C for 00:30:00 , 80 °C for 00:02:00 then hold at Room temperature
32m
Cleaving and dA-tailing target DNA
Cleaving and dA-tailing target DNA
35m
35m
Thaw the dATP tube, vortex to mix thoroughly, and place on ice
2m
Dilute dATP to concentration 10 millimolar (mM) . In a 0.2 ml thin-walled PCR tube, make a 10 millimolar (mM) dATP solution by adding 1 µL of the 100 millimolar (mM) dATP stock to 9 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 40 µL dephosphorylated DNA sample, add:
| A | B | |
| Reagent | Volume | |
| Dephosphorylated genomic DNA sample (Section 2) | 40 µl | |
| Cas9 RNPs (Section 1) | 15 µl | |
| 10 mM dATP | 1.5 µl | |
| Taq polymerase | 1 µl | |
| Total | 57.5 µl |
5m
Carefully mix the contents of the tube by gentle inversion, then spin down and place the tube in the thermal cycler
30s
Using the thermal cycler, incubate at 37 °C for 15-60 (00:15:00 are recommended) minutes, then 72 °C for 00:10:00 and hold at 4 °C or return to the tube to ice
25m
Adapter ligation
Adapter ligation
25m
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:
| A | B | |
| Reagent | Volume | |
| Ligation Buffer (LNB) | 25 µl | |
| Nuclease-free water | 5 µl | |
| NEBNext Quick T4 DNA Ligase | 12.5 µl | |
| Adapter Mix (AMX)* | 5 µl | |
| Total | 47.5 µl |
* The Adapter Mix (AMX) must be added last and immediately before the ligation step
2m
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
Add 20 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 (27.5 µL ) of the adapter ligation mix to the cleaved and dA-tailed sample, to yield a 105 µL ligation mix
1m
Mix gently by flicking the tube, and spin down
30s
Incubate the reaction for 00:20:00 at Room 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
1h
Add 1 volume (105 µL ) of TE (8.0 ) to the ligation mix. Mix gently by flicking the tube
1m
Add 0.3x volume (63 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 00:10:00 at Room 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
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant
2m
Wash the beads by adding either 250 µL Long Fragment Buffer (LFB) or 250 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
Repeat the previous step go to step #41
2m
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow drying for 00:00:30 , but do not dry the pellet to the point of cracking
30s
Remove the tube from the magnetic rack and resuspend the pellet in 13 µL Elution Buffer (EB). Incubate for 00:10:00 at Room temperature
Note
For fragments > 30 kb, we recommend increasing the elution time to 00:30:00
10m
Pellet the beads on a magnet until the eluate is clear and colorless
1m
Remove and retain 12 µL of eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
1m
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
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/s13059-015-0846-3Step 16
Boas Pucker. Plant DNA extraction and preparation for ONT sequencing
https://protocols.io/view/plant-dna-extraction-and-preparation-for-ont-seque-bcvyiw7w