Letter | Published:

Saturation editing of genomic regions by multiplex homology-directed repair

Nature volume 513, pages 120123 (04 September 2014) | Download Citation

Abstract

Saturation mutagenesis1,2—coupled to an appropriate biological assay—represents a fundamental means of achieving a high-resolution understanding of regulatory3 and protein-coding4 nucleic acid sequences of interest. However, mutagenized sequences introduced in trans on episomes or via random or “safe-harbour” integration fail to capture the native context of the endogenous chromosomal locus5. This shortcoming markedly limits the interpretability of the resulting measurements of mutational impact. Here, we couple CRISPR/Cas9 RNA-guided cleavage6 with multiplex homology-directed repair using a complex library of donor templates to demonstrate saturation editing of genomic regions. In exon 18 of BRCA1, we replace a six-base-pair (bp) genomic region with all possible hexamers, or the full exon with all possible single nucleotide variants (SNVs), and measure strong effects on transcript abundance attributable to nonsense-mediated decay and exonic splicing elements. We similarly perform saturation genome editing of a well-conserved coding region of an essential gene, DBR1, and measure relative effects on growth that correlate with functional impact. Measurement of the functional consequences of large numbers of mutations with saturation genome editing will potentially facilitate high-resolution functional dissection of both cis-regulatory elements and trans-acting factors, as well as the interpretation of variants of uncertain significance observed in clinical sequencing.

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Accessions

Primary accessions

Sequence Read Archive

Data deposits

Sequence data used for this analysis are available in SRA under accession number SRP044126.

References

  1. 1.

    , & Fine structure genetic analysis of a beta-globin promoter. Science 232, 613–618 (1986)

  2. 2.

    & High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244, 1081–1085 (1989)

  3. 3.

    et al. High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nature Biotechnol. 27, 1173–1175 (2009)

  4. 4.

    et al. High-resolution mapping of protein sequence-function relationships. Nature Methods 7, 741–746 (2010)

  5. 5.

    & Strategies and applications of in vitro mutagenesis. Science 229, 1193–1201 (1985)

  6. 6.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

  7. 7.

    , & The transience of transient overexpression. Nature Methods 10, 715–721 (2013)

  8. 8.

    , & ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405 (2013)

  9. 9.

    et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013)

  10. 10.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013)

  11. 11.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

  12. 12.

    et al. A BRCA1 nonsense mutation causes exon skipping. Am. J. Hum. Genet. 62, 713–715 (1998)

  13. 13.

    & CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnol. 32, 347–355 (2014)

  14. 14.

    et al. Quantitative evaluation of all hexamers as exonic splicing elements. Genome Res. 21, 1360–1374 (2011)

  15. 15.

    , & GC content around splice sites affects splicing through pre-mRNA secondary structures. BMC Genomics 12, 90 (2011)

  16. 16.

    et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nature Biotechnol. 30, 265–270 (2012)

  17. 17.

    et al. MutPred Splice: machine learning-based prediction of exonic variants that disrupt splicing. Genome Biol. 15, R19 (2014)

  18. 18.

    , , & Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014)

  19. 19.

    et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnol. 31, 827–832 (2013)

  20. 20.

    et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 2281–2308 (2013)

  21. 21.

    et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009)

  22. 22.

    et al. A general framework for estimating the relative pathogenicity of human genetic variants. Nature Genet. 46, 310–315 (2014)

  23. 23.

    , , & Structure-function analysis of yeast RNA debranching enzyme (Dbr1), a manganese-dependent phosphodiesterase. Nucleic Acids Res. 33, 6349–6360 (2005)

  24. 24.

    , , , & Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cell. Biol. 18, 93–101 (1998)

  25. 25.

    et al. Transient cold shock enhances zinc-finger nuclease-mediated gene disruption. Nature Methods 7, 459–460 (2010)

  26. 26.

    et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nature Methods 8, 753–755 (2011)

  27. 27.

    et al. FLASH assembly of TALENs for high-throughput genome editing. Nature Biotechnol. 30, 460–465 (2012)

  28. 28.

    Genome engineering with targetable nucleases. Annu. Rev. Biochem. 83, 409–439 (2014)

  29. 29.

    et al. DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells. Nature Chem. Biol. 10, 623–625 (2014)

  30. 30.

    , , & Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence. Proc. Natl Acad. Sci. USA 107, 9158–9163 (2010)

  31. 31.

    , & Binding of DAZAP1 and hnRNPA1/A2 to an exonic splicing silencer in a natural BRCA1 exon 18 mutant. Mol. Cell. Biol. 28, 3850–3860 (2008)

  32. 32.

    , , & PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30, 614–620 (2014)

  33. 33.

    & Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

  34. 34.

    & Amino acid substitution matrices from protein blocks. Proc. Natl Acad. Sci. USA 89, 10915–10919 (1992)

  35. 35.

    et al. A method and server for predicting damaging missense mutations. Nature Methods 7, 248–249 (2010)

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Acknowledgements

We thank F. Zhang and his laboratory for the CRISPR/Cas9 backbone constructs used in this study and G. Church and his laboratory for providing reagents used to establish CRISPR/Cas9 editing techniques in our lab. We also thank members of the Shendure laboratory for helpful discussions and D. Prunkard for assistance with FACS. This work was supported by the National Institutes of Health (DP1HG007811 to J.S.) and the UW Medical Scientist Training Program (G.M.F. and J.K.).

Author information

Author notes

    • Gregory M. Findlay
    •  & Evan A. Boyle

    These authors contributed equally to this work.

Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA

    • Gregory M. Findlay
    • , Evan A. Boyle
    • , Ronald J. Hause
    • , Jason C. Klein
    •  & Jay Shendure

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Contributions

The project was conceived and designed by G.M.F. and J.S. G.M.F. and E.A.B. performed experiments. E.A.B. and R.J.H. performed data analysis and generated data figures. G.M.F. generated schematic figures. G.M.F., E.A.B., R.J.H. and J.S. wrote the manuscript. J.C.K. assisted G.M.F to establish genome editing techniques in the laboratory.

Competing interests

We have filed a provisional patent application on the method.

Corresponding authors

Correspondence to Gregory M. Findlay or Jay Shendure.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains a Supplementary discussion of the potential sources of noise in the experiments (Supplementary Note 1) and a discussion of potential future applications of the methods presented in the paper (Supplementary Note 2).

Excel files

  1. 1.

    Supplementary Table 1

    This table contains a list of oligonucleotide sequences used in this study.

  2. 2.

    Supplementary Table 2

    This table contains enrichment scores from the BRCA1 exon 18 hexamer experiment.

  3. 3.

    Supplementary Table 3

    This table contains effect sizes from the BRCA1 whole exon 18 SNV experiment.

  4. 4.

    Supplementary Table 4

    This table contains enrichment scores from the DBR1 experiment.

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DOI

https://doi.org/10.1038/nature13695

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