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Improved HTGTS for CRISPR/Cas9 Off-target Detection Protocol

Experiment Summary

Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a "bait" DNA double-stranded break (DSB) to capture genome-wide "prey" DNA DSBs. Here, we described an improved HTGTS (iHTGTS) method, in which size-selection beads were used to enhance reaction efficiency and a new primer system was designed to be compatible with Illumina Hiseq sequencing. Compared with LAM-HTGTS, iHTGTS is lower cost and has much higher sensitivity for off-target detection in HEK293T, K562, U2OS and HCT116 cell lines. So we believe that iHTGTS is a powerful method for comprehensively assessing Cas9 off-target effect.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml tube
  3. 0.22 μm syringe filter
  4. 200 μl PCR tubes
  5. Agarose
  6. 1 kb DNA plus ladder
  7. Streptavidin C1 beads
  8. AMPure XP beads
  9. Protease K
  10. FastPfu
  11. dNTPs
  12. AxyPrep MAG PCR Clean-Up
  13. NaCl
  14. EDTA
  15. Tris
  16. PEG 8000
  17. T4 ligase
  18. EasyTaq
  19. Gel extraction kit
  20. 75% Ethanol
  21. Isopropyl
  22. EDTA-Na2·2H2O
  23. NaOH
  24. HCl
  25. SDS
  26. TAE
  27. Proteinase K
  28. 5 M NaCl
  29. 0.5 M EDTA (pH 8.0)
  30. 1 M Tris-HCl (pH 7.4)
  31. Cell lysis buffe
  32. TE buffer
  33. 50% (wt/vol) PEG 8000
  34. 2x B&W buffer
  35. Annealing buffer
  36. 50 mM bridge adapter

Equipment

  1. Foam floating tube rack
  2. 8-well magnet stand
  3. Pipettes
  4. Thermomixer C
  5. PCR Thermal Cyclers
  6. Covaris
  7. Centrifuge
  8. NanoDrop
  9. Incubator
  10. Autoclave

Procedure

A. Design the primers used for iHTGTS

Choose Cas9-generated on-target site DSB as the "bait" to capture other genome-wide "prey" DSBs. To be compatible with 2 x 150 bp Hiseq sequencing, biotinylated primer for LAM-PCR was designed to bind 150 bp upstream of Cas9 binding site; nested primer was designed annealling to the downstream of the biotinylated primer about 90 bp away from the cut site.

Primer design for iHTGTS.Fig. 1 Primer design for iHTGTS.

The sequences of primers and bridge adapters.Fig. 2 The sequences of primers and bridge adapters.

B. Extract Genomic DNA (gDNA)

  1. Forty-eight hours after transfecting HEK293T cells with the Cas9 plasmids, collect 107 transfected HEK293T cells (we have also tried K562, HCT116 and U2OS, all work well) in a 1.5 ml tube and add 500 µl cell lysis buffer. Incubate the tube in Thermomixer at 56 °C, 500 x g for 10-18 h.
  2. Add 500 µl isopropyl and mix thoroughly till you can see a white flocculent DNA pellet.
  3. Using a pipet to transfer the pellet into another 1.5 ml tube with 1 ml 70% ethanol. Centrifuge at 13,000 x g for 5 min.
  4. Discard the supernatant. Centrifuge again and deplete residual 70% ethanol. Add 500 µl TE and incubate the tube in Thermomixer at 56 °C, 500 x g for at least 2 h.
  5. Quantify the DNA using NanoDrop. The A260/A280 should be higher than 1.8.
  6. Recommend 20 µg gDNA for iHTGTS library construction.

C. Fragment gDNA by sonication

  1. Add 20 µg gDNA into a PCR tube. Set Covaris with the following parameters: PIP = 50 watts, DF = 30%, CPB = 200, Time = 60 s.
  2. After sonication, take 200 ng DNA for 1% agarose page. The range of the DNA smear should be at 0.2-2 kb with a peak at 0.75 kb.

DNA smear pattern after sonication.Fig. 3 DNA smear pattern after sonication.

D. LAM-PCR

  1. Set up the reaction in 4 x 50 µl PCR tubes as following (DNA template for each PCR reaction can be 1-10 µg):

    2. Set up the PCR program

  2. Set up the PCR program:
    95 °C for 2 min
    [95 °C for 30 s, 58 °C for 30 s, 72 °C for 1.5 min] (80 cycles)
    72 °C for 2 min
    10 °C forever
  3. Deplete surplus biotin primer using AMPure XP beads
    a) Add 50 µl AMPure XP beads into each PCR tube, mix gently and incubate the tubes at RT for 5 min.
    b) Put the tubes on an 8-well magnet stand for 5 min.
    c) Remove the supernatant, add 200 µl 70% ethanol. After standing for 30 s, remove the supernatant.
    d) Repeat the Step D3c.
    e) Add 50 µl dH2O, mix the beads completely using a pipette and incubate at RT for 2 min.
    f) Put the tubes on the magnet for 2 min and pool the supernatants (about 200 µl) into a new 1.5 ml tube.

E. Streptavidin beads binding

  1. Add 50 µl 5 M NaCl, 2.5 µl 0.5 M EDTA into the PCR product from the last step. Add 30 µl streptavidin beads and rotate for 4 h at RT. (The streptavidin beads should be washed twice with 1x B&W buffer before use)
  2. Put the beads against the 1.5 ml tube magnet stand and remove the supernatant. Wash the beads three times each using 400 µl 1x B&W buffer.
  3. Wash the beads with 400 µl dH2O and then resuspend the beads in 42.4 µl dH2O.

F. On-beads ligation for bridge adapter

Set the reaction in a 1.5 ml tube in a rotator and ligate overnight at RT.

Set the reaction in a 1.5 ml tube in a rotator and ligate overnight at RT.

G. Nested PCR

  1. Add 80 µl 2x B&W buffer and 160 µl 1x B&W buffer. Put the beads against the 1.5 ml tube magnet stand and remove the supernatant. Wash the beads using 400 µl 1x B&W buffer three times and 400 µl dH2O once. Resuspend the beads in 80 µl dH2O.
  2. Set up the PCR reaction in 2x PCR tubes as following:

    3. Set up the PCR program:

  3. Set up the PCR program:
    95 °C for 5 min
    [95 °C for 1 min, 58 °C for 30 s, 72 °C for 1 min] (15 cycles)
    72 °C for 10 min
    10 °C forever
  4. Recycle the PCR products using AMpure XP beads as described in Step D3. Elute the PCR products with 35 µl dH2O for each PCR tube. Gather the PCR products together and measure the concentration.

Enzyme Blocking (Optional)

  1. Add 8 µl 10x enzyme buffer, 10 U blocking enzyme, incubate at 37 °C for 1 h or longer.
  2. Purify the DNA with GeneJET column, elute with 70 µl dH2O, and check the concentration.

H. Tagged PCR

  1. Set up the PCR reaction in 2 x PCR tubes as following:

    2. Set up the PCR program

  2. Set up the PCR program:
    95 °C for 3 min
    [95 °C for 20 s, 60 °C for 30 s, 72 °C for 1 min] (10-15 cycles)
    72 °C for 5 min
    10 °C forever

I. Purified PCR products

Pool the DNA together, run all the DNA on 1% agarose gel in TAE buffer, cut products between 500-900 bp, purify through a Gel extraction column, elute with 30 µl dH2O twice. Now the PCR product is ready for Hiseq sequencing.

Purified PCR products

Related Products and Services at Creative Biogene

LAM PCR Service

CRISPR Off-Target Analysis Service

* For research use only. Not intended for any clinical use.
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