Advertisement

Tracking CRISPR’s Footprints

  • Lin Lin
  • Yonglun LuoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1961)

Abstract

The programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and CRISPR-Cas9-derived gene editing and manipulation tools have revolutionized biomedical research over the past few years. One important category of assisting technologies in CRISPR gene editing is methods used for detecting and quantifying indels (deletions or insertions). These indels are caused by the repair of CRISPR-Cas9-introduced DNA double-stranded breaks (DBSs), known as CRISPR’s DNA cleavage footprints. In addition, CRISPR-Cas9 can also leave footprints to the DNA without introducing DSBs, known as CRISPR’s DNA-binding footprints. The indel tracking methods have contributed greatly to the improvement of CRISPR-Cas9 activity and specificity. Here, we review and discuss strategies developed over that past few years to track the CRISPR’s footprints, their advantages, and limitations.

Key words

CRISPR Cas9 Indels DSB Indel frequency Off-target 

Notes

Acknowledgments

L.L. is supported by grants from the Lundbeck Foundation. Y.L is supported by BGI-Shenzhen, BGI-Qingdao, and grants from the Shenzhen Sanming Medical Project. We thank the whole team of Lars Bolund Institute of Regenerative Medicine (LBI), BGI, for their work and assistance on the CRISPR technologies, and especially Jun Wang from LBI for assistance with preparing Fig. 1. Y.L. is also supported by the Guangdong Provincial Key Laboratory of Genome Read and Write (No. 2017B030301011).

Disclaimer Statement: The views expressed in this article are the personal views of the author and may not be understood or quoted as being made on behalf of or reflecting the position of the Lars Bolund Institute of Regenerative Medicine, BGI, or one of its working parties.

References

  1. 1.
    Chang HHY et al (2017) Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol 18(8):495–506PubMedCrossRefGoogle Scholar
  2. 2.
    McVey M, Lee SE (2008) MMEJ repair of double-strand breaks (director’s cut): deleted sequences and alternative endings. Trends Genet 24(11):529–538PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Ceccaldi R et al (2016) Repair pathway choices and consequences at the double-strand break. Trends Cell Biol 26(1):52–64PubMedCrossRefGoogle Scholar
  4. 4.
    Lahue RS et al (1989) DNA mismatch correction in a defined system. Science 245(4914):160–164PubMedCrossRefGoogle Scholar
  5. 5.
    Lindahl T (1974) An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc Natl Acad Sci U S A 71(9):3649–3653PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Sancar A, Rupp WD (1983) A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region. Cell 33(1):249–260PubMedCrossRefGoogle Scholar
  7. 7.
    Mehta A, Haber JE (2014) Sources of DNA double-strand breaks and models of recombinational DNA repair. Cold Spring Harb Perspect Biol 6(9):a016428PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6(6):507–512PubMedCrossRefGoogle Scholar
  9. 9.
    Epinat JC et al (2003) A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res 31(11):2952–2962PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Arnould S et al (2007) Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J Mol Biol 371(1):49–65PubMedCrossRefGoogle Scholar
  11. 11.
    Boch J et al (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326(5959):1509–1512PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Jinek M et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Mali P et al (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Barrangou R, Doudna JA (2016) Applications of CRISPR technologies in research and beyond. Nat Biotechnol 34(9):933–941PubMedCrossRefGoogle Scholar
  16. 16.
    Yeung AT et al (2005) Enzymatic mutation detection technologies. BioTechniques 38(5):749–758PubMedCrossRefGoogle Scholar
  17. 17.
    Zhu X et al (2014) An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Sci Rep 4:6420PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Liu C et al (2017) Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J Control Release 266:17–26PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Li L et al (2018) Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities. Biomaterials 171:207–218PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Senis E et al (2014) CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol J 9(11):1402–1412PubMedCrossRefGoogle Scholar
  21. 21.
    Schmidt F, Grimm D (2015) CRISPR genome engineering and viral gene delivery: a case of mutual attraction. Biotechnol J 10(2):258–272PubMedCrossRefGoogle Scholar
  22. 22.
    Ehrke-Schulz E et al (2016) Quantification of designer nuclease induced mutation rates: a direct comparison of different methods. Mol Ther Methods Clin Dev 3:16047PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Sentmanat MF et al (2018) A survey of validation strategies for CRISPR-Cas9 editing. Sci Rep 8(1):888PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    van Overbeek M et al (2016) DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks. Mol Cell 63(4):633–646PubMedCrossRefGoogle Scholar
  25. 25.
    Brinkman EK et al (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42(22):e168PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Brinkman EK et al (2018) Easy quantification of template-directed CRISPR/Cas9 editing. Nucleic Acids Res 46(10):e58PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Lin L et al (2017) Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells. J Biotechnol 247:42–49PubMedCrossRefGoogle Scholar
  28. 28.
    Jensen KT et al (2017) Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency. FEBS Lett 591(13):1892–1901PubMedCrossRefGoogle Scholar
  29. 29.
    Dehairs J et al (2016) CRISP-ID: decoding CRISPR mediated indels by Sanger sequencing. Sci Rep 6:28973PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Yang Z et al (2015) Fast and sensitive detection of indels induced by precise gene targeting. Nucleic Acids Res 43(9):e59PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Ramlee MK et al (2015) High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis. Sci Rep 5:15587PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    D'Agostino Y et al (2016) A rapid and cheap methodology for CRISPR/Cas9 zebrafish mutant screening. Mol Biotechnol 58(1):73–78PubMedCrossRefGoogle Scholar
  33. 33.
    Samarut E et al (2016) A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting analysis. BMC Genomics 17:547PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Findlay SD et al (2016) A digital PCR-based method for efficient and highly specific screening of genome edited cells. PLoS One 11(4):e0153901PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Pinheiro LB et al (2012) Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 84(2):1003–1011PubMedCrossRefGoogle Scholar
  36. 36.
    Kim H et al (2011) Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods 8(11):941–943PubMedCrossRefGoogle Scholar
  37. 37.
    Wen Y et al (2017) A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair. J Biol Chem 292(15):6148–6162PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Zhou Y et al (2016) Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system. Cell Mol Life Sci 73(13):2543–2563PubMedCrossRefGoogle Scholar
  39. 39.
    Fu L et al (2016) A simple and efficient method to visualize and quantify the efficiency of chromosomal mutations from genome editing. Sci Rep 6:35488PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Yang Y et al (2016) Highly efficient and rapid detection of the cleavage activity of Cas9/gRNA via a fluorescent reporter. Appl Biochem Biotechnol 180(4):655–667PubMedCrossRefGoogle Scholar
  41. 41.
    Ramakrishna S et al (2014) Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat Commun 5:3378PubMedCrossRefGoogle Scholar
  42. 42.
    Hussmann D et al (2017) IGF1R depletion facilitates MET-amplification as mechanism of acquired resistance to erlotinib in HCC827 NSCLC cells. Oncotarget 8(20):33300–33315PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Xue LJ, Tsai CJ (2015) AGEseq: analysis of genome editing by sequencing. Mol Plant 8(9):1428–1430PubMedCrossRefGoogle Scholar
  44. 44.
    Pinello L et al (2016) Analyzing CRISPR genome-editing experiments with CRISPResso. Nat Biotechnol 34(7):695–697PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Boel A et al (2016) BATCH-GE: batch analysis of next-generation sequencing data for genome editing assessment. Sci Rep 6:30330PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Qi LS et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173–1183PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Ran FA et al (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6):1380–1389PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Hilton IB et al (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33(5):510–517PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Vad-Nielsen J et al (2018) Simple method for assembly of CRISPR synergistic activation mediator gRNA expression array. J Biotechnol 274:54–57PubMedCrossRefGoogle Scholar
  50. 50.
    Xiong K et al (2017) RNA-guided activation of pluripotency genes in human fibroblasts. Cell Reprogram 19(3):189–198PubMedCrossRefGoogle Scholar
  51. 51.
    Gaudelli NM et al (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551(7681):464–471PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Kim D et al (2015) Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods 12(3):237–243. 1 p following 243PubMedCrossRefGoogle Scholar
  53. 53.
    Tsai SQ et al (2017) CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods 14(6):607–614PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Cameron P et al (2017) Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nat Methods 14(6):600–606PubMedCrossRefGoogle Scholar
  55. 55.
    Frock RL et al (2015) Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol 33(2):179–186PubMedCrossRefGoogle Scholar
  56. 56.
    Tsai SQ et al (2015) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33(2):187–197PubMedCrossRefGoogle Scholar
  57. 57.
    Wang X et al (2015) Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol 33(2):175–178PubMedCrossRefGoogle Scholar
  58. 58.
    Crosetto N et al (2013) Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods 10(4):361–365PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Smith C et al (2014) Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15(1):12–13PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Veres A et al (2014) Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell 15(1):27–30PubMedPubMedCentralCrossRefGoogle Scholar
  61. 61.
    Slaymaker IM et al (2016) Rationally engineered Cas9 nucleases with improved specificity. Science 351(6268):84–88PubMedCrossRefGoogle Scholar
  62. 62.
    Kleinstiver BP et al (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529(7587):490–495PubMedPubMedCentralCrossRefGoogle Scholar
  63. 63.
    Kuscu C et al (2014) Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol 32(7):677–683PubMedCrossRefGoogle Scholar
  64. 64.
    Lin L et al (2018) Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7(3):1–19PubMedCrossRefGoogle Scholar
  65. 65.
    Kosicki M et al (2017) Dynamics of indel profiles induced by various CRISPR/Cas9 delivery methods. Prog Mol Biol Transl Sci 152:49–67PubMedCrossRefGoogle Scholar
  66. 66.
    Kosicki M et al (2018) Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36:765–771PubMedPubMedCentralCrossRefGoogle Scholar
  67. 67.
    Haeussler M et al (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17(1):148PubMedPubMedCentralCrossRefGoogle Scholar
  68. 68.
    Shou J et al (2018) Precise and predictable CRISPR chromosomal rearrangements reveal principles of Cas9-mediated nucleotide insertion. Mol Cell 71(4):498–509.e4PubMedCrossRefGoogle Scholar
  69. 69.
    Henrik Devitt Møller LL, Xi X, Petersen TS, Huang J, Yang L, Kjeldsen E, Jensen UB, Zhang X, Liu X, Xun X, Wang J, Yang H, Church GM, Bolund L, Regenberg B, Luo Y (2018) CRISPR-C: circularization of genes and chromosome by CRISPR in human cells. Nucleic Acids Res. https://doi.org/10.1093/nar/gky767

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiomedicineAarhus UniversityAarhusDenmark
  2. 2.BGI-ShenzhenShenzhenChina
  3. 3.Guangdong Provincial Key Laboratory of Genome Read and WriteShenzhenChina
  4. 4.BGI-QingdaoQingdaoChina

Personalised recommendations