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Genome-Wide CRISPR Off-Target DNA Break Detection by the BLISS Method

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2162)

Abstract

Clustered regularly interspaced palindromic repeat (CRISPR) systems are revolutionizing many areas of biology and medicine, where they are increasingly utilized as therapeutic tools for correcting disease-causing mutations. From a clinical perspective, unintended off-target (OT) DNA double-strand break (DSB) induction by CRISPR nucleases represents a major concern. Therefore, in recent years considerable effort has been dedicated to developing methods for assessing the OT activity of CRISPR nucleases, which in turn can be used to guide engineering of nucleases with minimal OT activity. Here we describe a detailed protocol for quantifying OT DSBs genome-wide in cultured cells transfected with CRISPR enzymes, based on the breaks labeling in situ and sequencing (BLISS) method that we have previously developed. CRISPR-BLISS is versatile and scalable, and allows assessment of multiple guide RNAs in different cell types and time points following cell transfection or transduction.

Key words

CRISPR Off-targets DNA double-strand breaks BLISS 

Notes

Acknowledgments

We thank Winston Yan and Feng Zhang from the Broad Institute for support in setting up CRISPR-BLISS.

References

  1. 1.
    Ran FA, Hsu PD, Wright J et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308.  https://doi.org/10.1038/nprot.2013.143CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ran FA, Cong L, Yan WX et al (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–191.  https://doi.org/10.1038/nature14299CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Paquet D, Kwart D, Chen A et al (2016) Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533:125–129.  https://doi.org/10.1038/nature17664CrossRefPubMedGoogle Scholar
  4. 4.
    Kwart D, Paquet D, Teo S, Tessier-Lavigne M (2017) Precise and efficient scarless genome editing in stem cells using CORRECT. Nat Protoc 12:329–334.  https://doi.org/10.1038/nprot.2016.171CrossRefPubMedGoogle Scholar
  5. 5.
    Kleinstiver BP, Pattanayak V, Prew MS et al (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490–495.  https://doi.org/10.1038/nature16526CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Akcakaya P, Bobbin ML, Guo JA et al (2018) In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature 561:416–419.  https://doi.org/10.1038/s41586-018-0500-9CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Tanenbaum ME, Gilbert LA, Qi LS et al (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635–646.  https://doi.org/10.1016/j.cell.2014.09.039CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Cheng AW, Wang H, Yang H et al (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 23:1163–1171.  https://doi.org/10.1038/cr.2013.122CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Gilbert LA, Horlbeck MA, Adamson B et al (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159:647–661.  https://doi.org/10.1016/j.cell.2014.09.029CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Rock JM, Hopkins FF, Chavez A et al (2017) Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat Microbiol 2:1–9.  https://doi.org/10.1038/nmicrobiol.2016.274CrossRefGoogle Scholar
  11. 11.
    Thakore PI, Kabadi AM, Safi A et al (2015) Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods 12:1143–1149.  https://doi.org/10.1038/nmeth.3630CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Okada M, Kanamori M, Someya K et al (2017) Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells. Epigenet Chrom 10:1–17.  https://doi.org/10.1186/s13072-017-0129-1CrossRefGoogle Scholar
  13. 13.
    Fu Y, Rocha PP, Luo VM et al (2016) CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci. Nat Commun 7:1–8.  https://doi.org/10.1038/ncomms11707CrossRefGoogle Scholar
  14. 14.
    Ghosh D, Venkataramani P, Nandi S, Bhattacharjee S (2019) CRISPR–Cas9 a boon or bane: the bumpy road ahead to cancer therapeutics. Cancer Cell Int 19:1–10.  https://doi.org/10.1186/s12935-019-0726-0CrossRefGoogle Scholar
  15. 15.
    Lin Y, Cradick TJ, Brown MT et al (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res 42:7473–7485.  https://doi.org/10.1093/nar/gku402CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Tsai SQ, Zheng Z, Nguyen NT et al (2015) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33:187–198.  https://doi.org/10.1038/nbt.3117CrossRefPubMedGoogle Scholar
  17. 17.
    Newton MD, Taylor BJ, Driessen RPC et al (2019) DNA stretching induces Cas9 off-target activity. Nat Struct Mol Biol.  https://doi.org/10.1038/s41594-019-0188-z
  18. 18.
    Kuscu C, Arslan S, Singh R et al (2014) Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol 32:677–683.  https://doi.org/10.1038/nbt.2916CrossRefPubMedGoogle Scholar
  19. 19.
    O’Geen H, Henry IM, Bhakta MS et al (2015) A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res 43:3389–3404.  https://doi.org/10.1093/nar/gkv137CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kosicki M, Tomberg K, Bradley A (2018) Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36.  https://doi.org/10.1038/nbt.4192
  21. 21.
    Fu Y, Foden JA, Khayter C et al (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826.  https://doi.org/10.1038/nbt.2623CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Cameron P, Fuller CK, Donohoue PD et al (2017) Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nat Methods 14:600–606.  https://doi.org/10.1038/nmeth.4284CrossRefPubMedGoogle Scholar
  23. 23.
    Knight SC, Xie L, Deng W et al (2015) Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science (80) 350:823–826.  https://doi.org/10.1126/science.aac6572CrossRefGoogle Scholar
  24. 24.
    Kim D, Kim JS (2018) DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA. Genome Res 28:1882–1893.  https://doi.org/10.1101/gr.236620.118CrossRefGoogle Scholar
  25. 25.
    Chiarle R, Zhang Y, Frock RL et al (2011) Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 147:107–119.  https://doi.org/10.1016/j.cell.2011.07.049CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Frock RL, Hu J, Meyers RM et al (2015) Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol 33:179–188.  https://doi.org/10.1038/nbt.3101CrossRefPubMedGoogle Scholar
  27. 27.
    Wang X, Wang Y, Wu X et al (2015) Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol 33:175–179.  https://doi.org/10.1038/nbt.3127CrossRefPubMedGoogle Scholar
  28. 28.
    Crosetto N, Mitra A, Silva MJ et al (2013) Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods 10:361–365.  https://doi.org/10.1038/nmeth.2408CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Slaymaker IM, Yan WX, Gao L et al (2015) Rationally engineered Cas9 nucleases with improved specificity. Science (80) 351:84–88.  https://doi.org/10.1126/science.aad5227CrossRefGoogle Scholar
  30. 30.
    Duan J, Lu G, Xie Z et al (2014) Genome-wide identification of CRISPR/Cas9 off-targets in human genome. Cell Res 24:1009–1012.  https://doi.org/10.1038/cr.2014.87CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Wu X, Scott DA, Kriz AJ et al (2014) Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol 32:670–676.  https://doi.org/10.1038/nbt.2889CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Martin F, Sánchez-Hernández S, Gutiérrez-Guerrero A et al (2016) Biased and unbiased methods for the detection of off-target cleavage by CRISPR/Cas9: an overview. Int J Mol Sci 17.  https://doi.org/10.3390/ijms17091507
  33. 33.
    Kim D, Bae S, Park J et al (2015) Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods 12:237–243.  https://doi.org/10.1038/nmeth.3284CrossRefPubMedGoogle Scholar
  34. 34.
    Kim D, Kim S, Kim S et al (2016) Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex digenome-seq. Genome Res 26:406–415.  https://doi.org/10.1101/gr.199588.115.FreelyCrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Kim D, Kim D, Lee G et al (2019) Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat Biotechnol:1.  https://doi.org/10.1038/s41587-019-0050-1
  36. 36.
    Lensing SV, Marsico G, Hänsel-Hertsch R et al (2016) DSBCapture: in situ capture and sequencing of DNA breaks. Nat Methods 13:855–857.  https://doi.org/10.1038/nmeth.3960CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Canela A, Sridharan S, Sciascia N et al (2016) DNA breaks and end resection measured genome-wide by end sequencing. Mol Cell 63:898–911.  https://doi.org/10.1016/j.molcel.2016.06.034CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Canela A, Maman Y, Jung S et al (2017) Genome organization drives chromosome fragility. Cell 170:507–521.e18.  https://doi.org/10.1016/j.cell.2017.06.034CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Yan WX, Mirzazadeh R, Garnerone S et al (2017) BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks. Nat Commun 8:15058.  https://doi.org/10.1038/ncomms15058CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Gao L, Cox DBT, Yan WX et al (2017) Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol 35:789–792.  https://doi.org/10.1038/nbt.3900CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Tsai SQ, Nguyen NT, Malagon-Lopez J et al (2017) CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods 14:607–614.  https://doi.org/10.1038/nmeth.4278CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Lazzarotto CR, Nguyen NT, Tang X et al (2018) Defining CRISPR–Cas9 genome-wide nuclease activities with CIRCLE-seq. Nat Protoc 13:2615–2642.  https://doi.org/10.1038/s41596-018-0055-0CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Lee K, Zhang Y, Kleinstiver BP et al (2019) Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize. Plant Biotechnol J 17:362–372.  https://doi.org/10.1111/pbi.12982CrossRefPubMedGoogle Scholar
  44. 44.
    Tang PZ, Ding B, Peng L et al (2018) TEG-seq: an ion torrent-adapted NGS workflow for in cellulo mapping of CRISPR specificity. BioTechniques 65:259–267.  https://doi.org/10.2144/btn-2018-0105CrossRefPubMedGoogle Scholar
  45. 45.
    Mirzazadeh R, Kallas T, Bienko M, Crosetto N (2018) Genome – wide profiling of DNA double – strand breaks by the BLESS and BLISS methods. Methods Mol Biol:167–194Google Scholar
  46. 46.
    Iannelli F, Galbiati A, Capozzo I et al (2017) A damaged genome’s transcriptional landscape through multilayered expression profiling around in situ-mapped DNA double-strand breaks. Nat Commun 8:15656.  https://doi.org/10.1038/ncomms15656CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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Authors and Affiliations

  1. 1.Science for Life Laboratory (SciLifeLab), Research Division of Genome Biology, Department of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden

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