CRISPR/Cas9 Off-target Identification and Validation by GUIDE-Seq Protocol

In Silico off-Target Site Prediction by COSMID

1. Select the "Target Genome" for which off-target analysis should be performed.
2. Enter the gRNA sequence in the "Query Sequence" box.
3. Enter the PAM sequence of the Cas9 ortholog (NGG for SpCas9) and select the number of allowed indels and mismatches.
4. COSMID can perform PCR primer design for each off-target site identified. In the "PCR Primer Design Options," check "Perform Primer Design According to the Following Setting" and select "Illumina_250_paired."
5. Click "Download excel spreadsheet summary" to download the search results.
6. Order oligonucleotide primers for each off-target site identified.

Identification of Off-Target Sites Using GUIDE-seq

Genome Editing in CD34⁺ HSPCs for Integration of dsODN Tag at Cas9 Cut Sites (See Note 3 for GUIDE-seq in Other Cell Types)
1. Generate the 100 μM dsODN tag by combining the sense oligonucleotide (200 μM in 1×TE) and the anti-sense oligonucleotide (200 μM in 1×TE) and run the following annealing program on a thermocycler:
(a)Heat to 95 °C for 2 min.
(b)Ramp cool to 25 °C over 45 min.
(c)Hold at 12 °C.
2. Aliquot and store annealed dsODN tag at −20 °C. Avoid freeze thaw cycles.
3. Harvest CD34⁺ HSPCs and prepare 5 × 10⁵ cells for each electroporation reaction. Transfer cells to 15 mL tube and fill the tube to 15 mL with 1×PBS. Pellet the cells by spinning at 300 × g for 5 min.
4. Meanwhile, assemble the RNP complex. Carefully mix 1 μL sgRNA, 0.5 μL SpCas9, and 1 μL dsODN tag (20 μM) in a 0.2 mL PCR tube per electroporation. Incubate at room temperature for 15 min. Prepare the same amount dsODN tag for the mock-treated control sample as treated samples without RNP.
5. Aspirate as much medium and PBS as possible without disturbing the cell pellet.
6. Add 22 μL P3 solution per 5 × 105 CD34⁺ HSPCs for each electroporation and gently resuspend cells.
7. Transfer 20 μL of cells in P3 solution mixture to the 0.2 mL tube containing the RNP and dsODN tag.
8. Mix well by gentle flick. Transfer 20 μL of the mixture in one pipet into the center groove of 4D electroporation cuvette. Avoid bubbles.
9. Immediately perform electroporation using the CA137 program. Incubate cells for 10 min at room temperature.
10. Add 80 μL pre-warmed CD34⁺ HSPCs expansion medium into each cuvette. Transfer all cells from each cuvette to a 24-well plate containing 500 μL pre-warmed culture medium per well.
11. 48 h after electroporation, add 500 μL fresh pre-warmed medium to the wells.
12. After 4 days, harvest cells and extract genomic DNA.

Confirmation of dsODN Integration at the Target Site

1. Design primers that yield amplicon length between 400 and 500 bp.
2. Set up PCR using the genomic DNA of cells treated with RNP and from mock-treated cells for use as controls.
3. Send purified PCR products for Sanger sequencing using the forward or reverse primer used for PCR amplification.
4. Go to Inference of CRISPR Editing (ICE) web-based tool that determines rates of CRISPR/Cas9 knockout and knockin editing.
5. Enter the Sample Label, 17–23 nt guide RNA sequences without PAM and the Donor Sequence. The donor sequences should contain the 34-bp dsODN Tag sequence inserted at the cut site in forward or reverse orientation.
6. Upload a chromatogram file for both a Control File (mock-treated cells) and an Experiment File (RNP and dsODN tag treated cells).
7. Confirm targeted integration of dsODN Tag in forward or reverse orientation.

Quantification and Shearing of Genomic DNA

1. Determine the concentration of extracted genomic DNA by fluorometric DNA quantification assay.
2. For each genomic DNA sample, prepare two tubes (anti-sense/ sense or +/−) of 1 μg DNA in a total volume of 140 μL 1× TE buffer.
3. Place 130 μL of each tube in a Covaris S220 microTube and shear for 25 s on a M220 Covaris following manufacturer's instructions. Run settings should be:
(a)Average incident power = 10 W.
(b)Peak incident power = 50 W.
(c)Duty factor = 20%.
(d)Cycles/burst = 200 counts.
4. Move samples from microtube to a new plastic tube.
5. Run 5 μL of sheared DNA on a 1.5% agarose gel and verify that the bulk of the shearing is 500 bps.
6. Clean up samples with 1× magnetic beads (120 μL) and elute with 15 μL water (see Note 5 for detailed magnetic bead cleanup protocol). You may leave the samples with the beads on the magnet and move them when ready to add to tubes with end-repair master mix.

Y-Adapter Preparation

1. Prepare the following annealing reaction by mixing MiSeq Common Adapter with each of the A01–A16 oligos
2. Run the following annealing program on a thermocycler:
(a) Heat to 95 °C for 2 min.
(b) Ramp cool to 25 °C over 45 min.
(c) 4 °C hold.

End Repair

1. Make the following master mix at 1.2× number of samples and dispense 8.5 μL to each PCR tube or well
2. Add 14 μL of water eluted samples to tubes containing the master mix. You should have two PCR tubes for each sample, one sense and one anti-sense.
3. Mix with pipet set at 22 μL.
4. Run the following on a thermocycler with heated lid turned off:
(a) 12 °C for 15 min.
(b) 37 °C for 15 min.
(c) 72 °C for 15 min.
(d) 4 °C hold.

Adapter Ligation

1. To new PCR tubes, add 1.0 μL of one barcode of annealed A01–A16 oligos. Use the same barcode for the +/− strands of the same sample.
2. Add 2.0 μL T4 DNA ligase to each tube.
3. Add 22.5 μL of the end-repaired sample. Mix by pipetting, vortex, quick spin.
4. Run the following on a thermocycler with heated lid turned off:
(a) 16 °C for 30 min.
(b) 22 °C for 30 min.
(c) 4 °C hold.
5. Clean up samples with 0.9× magnetic beads (22.95 μL). Elute with 22 μL water.

Amplification of dsTag Integrated Sites

1.Make the following master mix for PCR1. Prepare two PCR1 reactions for each sample using GSP1+ or GSP1- primer. Add sufficient volume of all reagents for the appropriate number of PCR1 reactions. Only one GSP1 primer is used per PCR1 reaction. Dispense 20 μL master mix to each PCR tube.
2.Add 10 μL of adapter ligated sample prepared in 3.2.5 (f) to 20 μL PCR1 reaction. Make sure to set up two PCR1 reactions (GSP1+ or GSP1-) for each sample. Mix by pipetting, vortex, quick spin.
3. Run the following program on a thermocycler with heated lid on:
(a) 95 °C for 5 min.
(b) (95 °C for 30 s, 70 °C (−1 °C/cycle) for 2 min., 72 °C for 30 s) 15 cycles.
(c) (95 °C for 30 s, 55 °C for 1 min, 72 °C for 30 s) 10 cycles.
(d) 72 °C for 5 min.
(e) 4 °C hold.
4. Clean up samples with 1.2× magnetic beads (36 μL) and elute with 15 μL water (see Note 5).

Adaptor Labeling of Amplicons Above

1. Make the following master mix for PCR. Prepare two PCR reactions for each sample using GSP2+ or GSP2- primer. Add sufficient volume of all reagents for the appropriate number of PCR reactions above. Only one GSP2 primer is used per PCR reaction
2. Dispense 13.5 μL to each PCR tube or well.
3. Add 1.5 μL of 10 μM P7_# to each tube to make a unique pair with A01–A16.
4. Add 15 μL of the PCR1 sample to the PCR2 master mix. Make sure to use GSP2+ primer for the GSP1+ PCR1 mix and GSP2- primer for the GSP1- PCR1 mix. Mix by pipetting, vortex, quick spin.
5. Run the following program on a thermocycler with heated lid on:
(a) 95 °C for 5 min.
(b) (95 °C for 30 s, 70 °C (−1 °C/cycle) for 2 min, 72 °C for 30 s) 15 cycles.
(c) (95 °C for 30 s, 55 °C for 1 min, 72 °C for 30 s) 10 cycles.
(d) 72 °C for 5 min.
(e) 4 °C hold.
6. Clean up samples with 0.7× magnetic beads (36 μL) and elute with 30 μL TE.

Illumina Sequencing

1. Thaw cartridge from MiSeq Reagent Kit v2 (300-cycles) >1.5 h prior to use in a room temperature water bath and thaw buffer HT1 at 4°C.
2. Dilute 5 μL of 4 nM pooled library with equal volume of freshly prepared 0.2N NaOH and vortex briefly. Centrifuge at 280 × g for 1 min and incubate at room temperature for 5 min.
3. Dilute denatured library to 20 pM by adding 990 μL of chilled buffer HT1.
4. Further dilute denatured library to 12.5 pM by mixing 225 μL prechilled HT1 and 375 μL of a 20 pM denatured library.
5. Invert to mix and store on ice until ready to load onto the MiSeq reagent cartridge.
6. Dilute each GUIDE-seq read2 and GUIDE-seq index primers to 0.5 μM with HT1.
7. Prepare MiSeq Sample Sheet according to the MiSeq Sample Sheet Quick Ref Guide (15028392 J).
8. Pierce the foil seal of position 17, 19, and 20 on the reagent cartridge with a clean 1 mL pipette.
9. Pipette 600 μL of the 12.5 pM denatured library to the position 17 designated as "Load Sample." Avoid buddle.
10. Pipette 600 μL of GUIDE-seq index1 primer to position 19 of the reagent cartridge.
11. Pipette 600 μL of GUIDE-seq read 2 primer to position 20 of the reagent cartridge.
12. Select "Sequence" on the software interface and follow the instruction on the screen.
13. Select the sample sheet containing the run specific information.
14. Rinse the flow cell with Milli-Q water. Dry with lint-free cleaning tissue.
15. Load the flow cell, cartridge, and PR2.
16. Sequence.

Reference:

1. Park S H, Lee C M, Bao G. Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells[M]//Stem Cell Assays: Methods and Protocols. New York, NY: Springer US, 2022: 281-306.