TITLE: Cas9-targeted Nanopore sequencing (CANS)
AUTHORS: Pavel Merkulov, Ilya Kirov

[DESCRIPTION]
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.

[STEPS]
SECTION: Preparing the Cas9 ribonucleoprotein complexes (RNPs)
11. Add water to get 11 µL

SECTION: Preparing the Cas9 ribonucleoprotein complexes (RNPs)
12. Heat and cool each sgRNAs to obtain pure monomers: 95 °C for 3 min , then cool to 95 Room temperature for 2 min 
 

SECTION: Preparing the Cas9 ribonucleoprotein complexes (RNPs)
13. To form Cas9 RNPs, assemble the components in the table in a 1.5 ml Eppendorf DNA LoBind tube in the following order: 
ReagentVolume (per one cleavege reaction)Cas9 5x buffer3Cas91gRNA (50ng/ul ~ 1pmol/ul) in 11 ul water11Total 15
 
SECTION: Preparing the Cas9 ribonucleoprotein complexes (RNPs)
14. Mix thoroughly by flicking the tube
SECTION: Preparing the Cas9 ribonucleoprotein complexes (RNPs)
15. Form the RNPs by incubating the tube at 95 Room temperature for 30 min, then return the RNPs on ice until required (proceed to the 'Dephosphorylating genomic DNA' section during this time)

SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
16. Transfer 1-10 μg (with 5 μg recommended) genomic DNA into 0.2 mL tubes.
 
ml thin-walled PCR tubes
SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
17. Adjust to 24 µLwith nuclease-free water

SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
18. Mix thoroughly by flicking the tube avoiding unwanted shearing
SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
19. Spin down briefly in a microfuge
SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
20. Mix the Quick calf intestinal alkaline phosphatase (CIP) in the tube by pipetting up and down. Ensure that it is at 4 Room temperature before use

SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
21. Assemble the following components in a clean 0.2 ml thin-walled PCR tube:
ReagentVolumeNEB CutSmart Buffer (10x)4 µlHMW genomic DNA (at ≥ 210 ng/µl)*24-30 µlWaterto get 34 µl total volumeTotal34 µl
 
SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
22. Mix gently by flicking the tube, and spin down
SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
23. Add 6 µLof CIP to the tube

SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
24. Mix gently by flicking the tube, and spin down
SECTION: Dephosphorylating genomic DNA (This step reduces background reads by removing 5’ phosphates from non-target DNA ends.)
25. Using a thermal cycler, incubate at 37 °C for 30 min, 80 °C for 2 min then hold at 20 Room temperature 

SECTION: Cleaving and dA-tailing target DNA
26. Thaw the dATP tube, vortex to mix thoroughly, and place on ice
SECTION: Cleaving and dA-tailing target DNA
27. 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

SECTION: Cleaving and dA-tailing target DNA
28. Spin down and place the tube of Taq polymerase on ice
SECTION: Cleaving and dA-tailing target DNA
29. To the PCR tube containing 40 µLdephosphorylated DNA sample, add: 
ReagentVolumeDephosphorylated genomic DNA sample (Step 2)40 µlCas9 RNPs (Step 1)15 µl10 mM dATP1.5 µlTaq polymerase1 µlTotal57.5 µl


SECTION: Cleaving and dA-tailing target DNA
30. Carefully mix the contents of the tube by gentle inversion, then spin down and place the tube in the thermal cycler
SECTION: Cleaving and dA-tailing target DNA
31. Using the thermal cycler, incubate at 37 °C for 15-60 (15 min are recommended) minutes, then 72 °C for 10 min and hold at 4 °C or return to the tube to ice
SECTION: Adapter ligation
32. 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:
 
ReagentVolumeLigation Buffer (LNB)25 µlNuclease-free water5 µlNEBNext Quick T4 DNA Ligase12.5 µlAdapter Mix (AMX)*5 µlTotal47.5 µl

 
 
SECTION: Adapter ligation
33. Mix by pipetting the above ligation mix thoroughly. Ligation Buffer (LNB) is very viscous, so the adapter ligation mix needs to be well-mixed
SECTION: Adapter ligation
34. 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

SECTION: Adapter ligation
35. Mix gently by flicking the tube, and spin down
SECTION: Adapter ligation
36. Incubate the reaction for 20 min at 4 Room temperature
 


SECTION: AMPure XP bead purification
37. Add 1 volume (105 µL) of TE (pH 8.0) to the ligation mix. Mix gently by flicking the tube

SECTION: AMPure XP bead purification
38. 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

SECTION: AMPure XP bead purification
39. Incubate the sample for 10 min at 4 Room temperature
 



SECTION: AMPure XP bead purification
40. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant
SECTION: AMPure XP bead purification
41. 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

SECTION: AMPure XP bead purification
42. Repeat the previous step   
SECTION: AMPure XP bead purification
43. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow drying for 30 s, but do not dry the pellet to the point of cracking

SECTION: AMPure XP bead purification
44. Remove the tube from the magnetic rack and resuspend the pellet in 13 µL Elution Buffer (EB). Incubate for 10 min at 4 Room temperature
 

SECTION: AMPure XP bead purification
45. Pellet the beads on a magnet until the eluate is clear and colorless
SECTION: AMPure XP bead purification
46. Remove and retain 12 µL of eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube

SECTION: AMPure XP bead purification
47. 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)
SECTION: Preparing the Cas9 ribonucleoprotein complexes (RNPs)
10. Combine equimolar amounts of sgRNAs for a targeted fragment in a single tube.
SECTION: In vitro transcription of sgRNAs
1. 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).
 

SECTION: In vitro transcription of sgRNAs
6. Combine following components for T7 in vitro transcription of your sgRNA:

 

SECTION: In vitro transcription of sgRNAs
2. Combine following components for sgRNA template synthesis:
 


SECTION: In vitro transcription of sgRNAs
7. Incubate your reaction at 37 °C for 120 min. The incubation time can also be extended up to 960 min (overnight) to obtain a higher sgRNA yield.

SECTION: In vitro transcription of sgRNAs
3. 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


SECTION: In vitro transcription of sgRNAs
4. 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).

SECTION: In vitro transcription of sgRNAs
5. 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).
8. Purify your sgRNA template with your system of choice.  We use a kit for the isolation of total RNA and microRNA.
9. Check the structure of synthesized sgRNA with agarose gel electrophoresis (the number of bands depends on the secondary sgRNA structure).