GridION: Protocol

V Q_CS9_revA_30Jan2025

FOR RESEARCH USE ONLY

1. Overview of the protocol

Introduction to the CRISPR-Cas protocol

The Cas9 Sequencing Kit (Q-SQK-CS9109) uses the CRISPR-Cas family of proteins which can be programmed to cut specific sequences using oligonucleotide-length RNA, facilitating genome editing and targeted sequencing.

In Cas9 targeted sequencing, we can selectively protect and deprotect ends using CRISPR-Cas and enrich for regions of interest from a given genomic background prior to long read nanopore sequencing of the native DNA sample.

This protocol describes how to carry out sequencing of genomic DNA using the Cas9 Sequencing Kit (Q-SQK-CS9109) with enrichment of specific genomic regions using CRISPR-Cas. You will prepare a target gene region by using gene probes, pooling four probes per region in equimolar quantities. Please refer to the supplementary information for more detail.

Figure 1. The biochemical steps used to prepare your DNA library using the Cas9 Sequencing Kit.

After DNA extraction, 5’ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands.
Cas9 ribonucleoprotein particles (RNPs), with bound crRNA and tracrRNA, are added to the genomic DNA. They then bind and cleave the Region of Interest (ROI).
dsDNA cleavage by Cas9 reveals blunt ends with ligatable 5’ phosphates.
All DNA in the samples is dA-tailed, which prepares the blunt ends for ligation.
Sequencing adapters are ligated primarily to Cas9 cut sites, which are both 3’ dA-tailed and 5’ phosphorylated. The library preparation is cleaned up to remove excess adapters using AMPure XP beads and resuspended in Elution Buffer. Non-target molecules are not removed. The subsequent library preparation is added to the flow cell for sequencing.

Compatibility of this protocol

This protocol should only be used in combination with:

Cas9 Sequencing Kit (Q-SQK-CS9109)
R9.4.1 (Q-FLO-MIN106) MinION Flow Cells
Flow Cell Priming Kit (Q-EXP-FLP002)

2. Equipment and consumables

材料

5 µg high molecular weight genomic DNA at >210 ng/µl (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly
Cas9 Sequencing Kit (Q-SQK-CS9109)

消耗品

S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
1.5 ml Eppendorf DNA LoBind tubes
0.2 ml 薄壁のPCRチューブ
Nuclease-free water (e.g. ThermoFisher, AM9937)
Agencourt AMPure XP beads (Beckman Coulter, A63881)

装置

小型遠心機
マグネットラック
ボルテックスミキサー
サーマルサイクラー
P1000 ピペット及びチップ
P200 ピペットとチップ
P100 ピペットとチップ
P20 ピペットとチップ
P10 ピペットとチップ
P2 ピペットとチップ
アイスバケツ（氷入り）
タイマー

オプション装置

Agilent Bioanalyzer (or equivalent)
Qubit蛍光光度計（またはQCチェックのための同等品）
Eppendorf 5424 centrifuge (or equivalent)

For this protocol, you will need 1–10 µg or 0.1–2 pmol high molecular weight genomic DNA (5 µg recommended at a concentration of >210 ng/μl ).

Cas9 Sequencing Kit contents (Q-SQK-CS9109):

Name	Abbreviation	Cap colour	No. of vials	Fill volume per vial (μl)
Adapter Mix	AMX	Green	2	40
Ligation Buffer	LNB	White	2	200
L fragment Buffer	LFB	Orange	2	1,800
S fragment Buffer	SFB	Clear	2	1,800
Loading Beads	LB	Pink	1	360
Elution Buffer	EB	Black	1	200
Sequencing Buffer	SQB	Red	1	300
SPRI Dilution Buffer	SDB	Brown cap, red label	1	1,200
T4 DNA Ligase	LIG	Brown cap, blue label	1	140
dATP	dATP	Brown cap, grey label	1	15
Reaction Buffer	RB	Brown cap, orange label	1	180
Phosphatase	PHOS	Brown cap, yellow label	1	50
TAQ Polymerase	TAQ	Brown cap, green label	1	15
Flush Tether	FLT	White cap, lilac label	1	200
Flush Buffer	FB	Blue	6	1,170

Wide-bore tips

Use wide-bore tips (or regular pipette tips with the narrow ends cut off) where possible to minimise shearing of long DNA.

3. Computer requirements and software

GridION IT requirements

The GridION device contains all the hardware required to control up to five sequencing experiments simultaneously and acquire the data. The device is further enhanced with high performance GPU technology for real-time basecalling. Read more in the GridION Q IT Requirements document in the Q system channel.

The sequencing software

The sequencing software controls the GridION, collects sequencing data in real-time and processes it into basecalls. The software can also demultiplex reads by barcode, and basecall/demultiplex data after a sequencing run has completed. You will be using the sequencing software for every assay you run.

For instructions on how to run the sequencing software on the GridION, please refer to the Q-Line sequencing software user guide.

4. Preparing the Cas9 ribonucleoprotein complexes (RNPs)

材料

Reaction Buffer (RB)

消耗品

S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
0.2 ml 薄壁のPCRチューブ
1.5 ml Eppendorf DNA LoBind tubes
Nuclease-free water (e.g. ThermoFisher, AM9937)

装置

サーマルサイクラー
Ice bucket with wet ice
P200 ピペットとチップ
P10 ピペットとチップ
P2 ピペットとチップ

Here, the Cas9 is loaded with crRNA and tracrRNA to form ribonucleoprotein complexes (RNPs) in preparation for the cleavage reaction.

A single crRNA or many crRNA probes may be used in a single cleavage reaction. You will need to validate any use of multiple probes.
The crRNA probes may also be pre-mixed as an off-catalogue request from IDT.
For example, you can use probes for the HTT gene, found here, as an individual experiment or in addition to other probes as an in-run control.
Store any unused crRNA probe mix at -80ºC with minimal freeze-thawing.

Reagent	Volume
Nuclease-Free Duplex Buffer (IDT)	8 µl
crRNA pool (100 µM, equimolar)	1 µl
tracrRNA (100 µM)	1 µl
Total	10 µl

We do not recommend storing and reusing the annealed mix.

Reagent	Volume
Nuclease-free water	79.2 µl
Reaction Buffer (RB)	10 µl
Annealed crRNA•tracrRNA pool (10 µM)	10 µl (Step 4, above)
HiFi Cas9 (62 µM)	0.8 µl
Total	100 µl

The above step yields an excess amount of RNPs, but 10 µl are carried forwards for each reaction into the next target cleavage step. Store any excess RNPs at 4ºC for up to a week.

Proceed to the next step (gDNA dephosphorylation) during the 30 min RNP incubation step.

5. Dephosphorylating genomic DNA

材料

5 µg high molecular weight genomic DNA at >210 ng/µl (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly
Phosphatase (PHOS)
Reaction Buffer (RB)

消耗品

1.5 ml Eppendorf DNA LoBind tubes
Nuclease-free water (e.g. ThermoFisher, AM9937)
0.2 ml 薄壁のPCRチューブ

装置

サーマルサイクラー
P100 ピペットとチップ
P10 ピペットとチップ

This step reduces background reads by removing 5’ phosphates from non-target DNA ends.

Transfer 5 μg genomic DNA into a 0.2 ml thin-walled PCR tube
Adjust to 24 μl with nuclease-free water
Mix thoroughly by flicking the tube, avoiding unwanted shearing
Spin down briefly in a microfuge

Reagent	Volume
Reaction Buffer (RB)	3 µl
HMW genomic DNA (at ≥210 ng/µl)	24 µl
Total	27 µl

Note: For an initial test, we recommend a 5 µg genomic DNA input. Target coverage scales linearly with input amount, so the input amount may be reduced accordingly if a lower output is acceptable.

6. Cleaving and dA-tailing target DNA

材料

Dephosphorylated gDNA from the previous step
crRNA-tracrRNA-Cas9 ribonucleoprotein complexes (RNPs) from the previous step
Taq Polymerase (TAQ)
dATP (dATP)

消耗品

1.5 ml Eppendorf DNA LoBind tubes
0.2 ml 薄壁のPCRチューブ

装置

サーマルサイクラー
Ice bucket with wet ice
ボルテックスミキサー
P100 ピペットとチップ
P20 ピペットとチップ
P10 ピペットとチップ
P2 ピペットとチップ

In this step, Cas9 RNPs (see 'Preparing the Cas9 ribonucleoprotein complexes') and Taq polymerase are added to the dephosphorylated genomic DNA sample.

This process cleaves target and dA-tails all available DNA ends in one step, activating the Cas9 cut sites for ligation.

Reagent	Volume
Dephosphorylated genomic DNA sample	30 µl
Cas9 RNPs	10 µl
dATP	1 µl
Taq Polymerase (TAQ)	1 µl
Total	42 µl

7. Adapter ligation

材料

Cleaved and dA-tailed DNA sample
Ligation Buffer (LNB)
Adapter Mix (AMX)
T4 Ligase (LIG)

消耗品

1.5 ml Eppendorf DNA LoBind tubes
Agencourt AMPure XP beads (Beckman Coulter™, A63881)

装置

Ice bucket with wet ice
ボルテックスミキサー
P1000 ピペット及びチップ
P100 pipette
P20 ピペットとチップ

Here, AMX adapters from the Cas Sequencing Kit (Q-SQK-CS9109) are ligated to the ends generated by Cas9 cleavage.

Reagent	Volume
Ligation Buffer (LNB)	20 µl
Nuclease-free water	3 µl
T4 Ligase (LIG)	10 µl
Adapter Mix (AMX)	5 µl
Total	38 µl

DNA precipitation

A white precipitate may form upon 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 indicate failure of ligation of the sequencing adapter to target molecule ends.

8. AMPure XP bead purification

材料

Long Fragment Buffer (LFB)
Elution Buffer (EB)
SPRI Dilution Buffer (SDB)

消耗品

Agencourt AMPure XP beads (Beckman Coulter™, A63881)
1.5 ml Eppendorf DNA LoBind tubes

装置

P1000 ピペット及びチップ
P200 ピペットとチップ
P20 ピペットとチップ
1.5 mlエッペンドルフチューブに最適のマグネット式ラック
Eppendorf 5424 centrifuge (or equivalent)

This step removes excess unligated adapters and other short DNA fragments, and concentrates and buffer-exchanges the library in preparation for sequencing.

Note: For longer targets, an extended incubation time may be beneficial.

Dispose of the pelleted beads

The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.

Library storage recommendations

We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short term storage. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes.

9. Priming and loading the MinION Flow Cell

材料

Flush Tether (FLT)
Flush Buffer (FB)
Sequencing Buffer (SQB)
Loading Beads (LB)

消耗品

1.5 ml Eppendorf DNA LoBind tubes
Nuclease-free water (e.g. ThermoFisher, AM9937)

装置

MinION Flow Cell
P1000 ピペット及びチップ
P100 ピペットとチップ
P20 ピペットとチップ
P10 ピペットとチップ

Press down firmly on the flow cell to ensure correct thermal and electrical contact.

Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.

Set a P1000 pipette to 200 µl
Insert the tip into the priming port
Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip

Note: Visually check that there is continuous buffer from the priming port across the sensor array.

The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

Reagent	Volume per flow cell
Sequencing Buffer (SQB)	37.5 µl
Loading Beads (LB), mixed immediately before use	25.5 µl
DNA library	12 µl
Total	75 µl

Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer (SQB) and Loading Beads (LB) because the fuel in the buffer will start to be consumed by the adapter.

Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

10. Data acquisition and basecalling

How to start sequencing

Follow the instructions in the Q-Line sequencing software user guide beginning from the "Set up and run an assay" section.

Device:

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Documentation

Nanopore Learning

Ligation sequencing gDNA - Cas9 enrichment (Q-SQK-CS9109) (Q_CS9_revA_30Jan2025)

Introduction to the protocol

Library preparation

Sequencing and data analysis

ドキュメントのバージョン

GridION: Protocol

V Q_CS9_revA_30Jan2025

Contents

Introduction to the protocol

Library preparation

Sequencing and data analysis

1. Overview of the protocol

Introduction to the CRISPR-Cas protocol

Compatibility of this protocol

2. Equipment and consumables

材料

消耗品

装置

オプション装置

For this protocol, you will need 1–10 µg or 0.1–2 pmol high molecular weight genomic DNA (5 µg recommended at a concentration of >210 ng/μl ).

Cas9 Sequencing Kit contents (Q-SQK-CS9109):

Wide-bore tips

3. Computer requirements and software

GridION IT requirements

The sequencing software

4. Preparing the Cas9 ribonucleoprotein complexes (RNPs)

材料

消耗品

装置

Here, the Cas9 is loaded with crRNA and tracrRNA to form ribonucleoprotein complexes (RNPs) in preparation for the cleavage reaction.

Pre-heat a thermal cycler to 95ºC.

Thaw an aliquot of Reaction Buffer (RB), mix by vortexing, and place on ice.

In an 1.5 ml Eppendorf DNA LoBind tube, pool the crRNA probes for each cleavage reaction by combining equal volumes of each crRNA probe, resuspended at 100 µM in TE (pH 7.5).

Anneal the pooled crRNAs with tracrRNA in Nuclease-Free Duplex Buffer by assembling the following in a 0.2 ml thin-walled PCR tube, as follows:

Mix gently by pipetting and spin down.

Heat the above reaction mix in a thermal cycler at 95ºC for 5 mins.

We do not recommend storing and reusing the annealed mix.

Remove the tube from the thermal cycler and allow it to cool to room temperature (18°C–23°C).

Spin down the tube to collect any liquid in the bottom.

To form Cas9 RNPs, assemble the components in the table in an 1.5 ml Eppendorf DNA LoBind tube in the stated order:

The above step yields an excess amount of RNPs, but 10 µl are carried forwards for each reaction into the next target cleavage step. Store any excess RNPs at 4ºC for up to a week.

Thoroughly mix the reaction by gently pipetting and briefly spin down.

Form the RNPs by incubating the tube at room temperature (18°C–23°C) for 30 minutes, then return the RNPs to ice until required.

Proceed to the next step (gDNA dephosphorylation) during the 30 min RNP incubation step.

5. Dephosphorylating genomic DNA

材料

消耗品

装置

This step reduces background reads by removing 5’ phosphates from non-target DNA ends.

Prepare the DNA in nuclease-free water.

Mix the Phosphatase (PHOS) in the tube by pipetting up and down. Ensure that it is at room temperature (18°C–23°C) before use.

Assemble the following components in a clean 0.2 ml thin-walled PCR tube:

Mix the components by gently flicking the tube and spin down.

Add 3 µl of PHOS to the tube.

Mix the components by gently flicking the tube and spin down.

Incubate in a thermal cycler at 37ºC for 10 minutes, then 80ºC for 2 minutes.

Remove the tube from the thermal cycler and allow to cool to room temperature (18°C–23°C).

6. Cleaving and dA-tailing target DNA

材料

消耗品

装置

In this step, Cas9 RNPs (see 'Preparing the Cas9 ribonucleoprotein complexes') and Taq polymerase are added to the dephosphorylated genomic DNA sample.

Thaw the dATP tube, vortex to mix thoroughly and place on ice.

Spin down and place the tube of Taq Polymerase (TAQ) on ice.

To the PCR tube containing 30 µl dephosphorylated DNA sample, add:

Carefully mix the contents of the tube by gentle inversion, then spin down and place the tube in the thermal cycler.

Incubate in the thermal cycler at 37ºC for 60 minutes, then 72ºC for 5 minutes, then hold at 4ºC or return the tube to ice.

7. Adapter ligation

材料

消耗品

装置

Here, AMX adapters from the Cas Sequencing Kit (Q-SQK-CS9109) are ligated to the ends generated by Cas9 cleavage.

Thaw the Ligation Buffer (LNB) at room temperature (18°C–23°C), spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.

Spin down the Adapter Mix (AMX) and place on ice.

Bring the AMPure XP beads to room temperature.

Carefully transfer the contents of the 0.2 ml thin-walled PCR tube to a fresh 1.5 ml Eppendorf DNA LoBind Tube using a wide-bore pipette tip.

Add 20 µl of the adapter ligation mix to the cleaved and dA-tailed sample. Mix the components slowly and carefully by pipetting. Briefly spin down to collect material at the bottom of the tube.