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Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci

Nature Genetics volume 49, pages 625–634 (2017)Cite this article

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Abstract

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

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Figure 1: Trait associations of the HBS1L-MYB intergenic region.
Figure 2: DNA Striker algorithm.
Figure 3: Pooled saturating-mutagenesis screening of the HBS1L-MYB region by using NGG- and NGA Cas9s and variants from 1000 Genomes haplotypes.
Figure 4: Mapping NGG- and NGA-restricted sgRNA dropout scores to genomic cleavage position identifies putative functional elements.
Figure 5: Trait-associated SNPs mark essential enhancer elements.
Figure 6: The HBS1L-MYB intergenic region contains highly repetitive genomic sequences.

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Acknowledgements

We thank Z. Herbert, M. Berkeley, and M. Vangala (Dana-Farber Cancer Institute Molecular Biology Core Facility) for sequencing, F. Lu at the HHMI Sequencing facility, and members at the Hematologic Neoplasia Flow Cytometry and the Flow Cytometry Core facilities at the Dana-Farber Cancer Institute for cell-sorting. We also thank J. Doench, M. Haeussler, J.-P. Concordet, R. Barretto, V. Sankaran, and J. Xu for helpful discussions. M.C.C. is supported by a National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) award (F30DK103359-01A1). L.P. is supported by a National Human Genome Research Institute (NHGRI) Career Development Award (K99HG008399). S.L. is funded by a Canadian Institutes of Health Research Banting Doctoral Scholarship. E.N.S. is supported by a Hematology Opportunities for the Next Generation of Research Scientists (HONORS) Award from the American Society of Hematology. G.C.Y. is supported by awards from the National Heart, Lung, and Blood Institute (NHLBI) (R01HL119099). G.L. is funded by the Canada Research Program, the Montreal Heart Institute Foundation, and the Canadian Institute of Health Research (MOP123382). A portion of the DNA genotyping was performed as part of the Biogen Sickle Cell Disease Consortium. D.E.B. is supported by NIDDK (K08DK093705, R03DK109232), NHLBI (DP2OD022716), the Burroughs Wellcome Fund, a Doris Duke Charitable Foundation Innovations in Clinical Research Award, an ASH Scholar Award, a Charles H. Hood Foundation Child Health Research Award, and a Cooley's Anemia Foundation Fellowship. S.H.O. is supported by an award from the NHLBI (P01HL032262) and an award from the NIDDK (P30DK049216, Center of Excellence in Molecular Hematology).

Author information

Author notes

  1. Daniel E Bauer and Stuart H Orkin: These authors jointly supervised this work.

Authors and Affiliations

  1. Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School,

    Matthew C Canver, Yuxuan Wu, Emily N Stern, Austen J Needleman, Diane D Chen, Partha Pratim Das, Mitchel A Cole, Jing Zeng, Daniel E Bauer & Stuart H Orkin

  2. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Matthew C Canver, Yuxuan Wu, Emily N Stern, Austen J Needleman, Diane D Chen, Partha Pratim Das, Mitchel A Cole, Jing Zeng, Daniel E Bauer & Stuart H Orkin

  3. Montreal Heart Institute, Université de Montréal, Montréal, Québec, Canada

    Samuel Lessard, Yann Ilboudo & Guillaume Lettre

  4. Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Luca Pinello

  5. Red Cell Genetic Disease Unit, Hôpital Henri-Mondor, Assistance Publique–Hôpitaux de Paris (AP-HP), UPeC, IMRB U955 Equipe no. 2, Créteil, France

    Frédéric Galactéros

  6. Department of Laboratory Medicine, Boston Children's Hospital, Boston, Massachusetts, USA

    Carlo Brugnara

  7. Department of Medicine, Sickle Cell Center, Augusta University, Augusta, Georgia, USA

    Abdullah Kutlar

  8. The Caribbean Institute for Health Research, University of the West Indies, Mona, Kingston, Jamaica.,

    Colin McKenzie & Marvin Reid

  9. Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, Tokyo, Japan

    Ryo Kurita

  10. Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Japan

    Yukio Nakamura

  11. Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

    Yukio Nakamura

  12. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA

    Guo-Cheng Yuan

  13. Howard Hughes Medical Institute, Boston, Massachusetts, USA

    Stuart H Orkin

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  1. Matthew C Canver
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Contributions

M.C.C., D.E.B., and S.H.O. conceived this study. M.C.C. developed the DNA Striker computational tool and performed computational analysis of degrees of PAM saturation. M.C.C., Y.W., E.N.S., A.J.N., D.D.C., P.P.D., M.A.C., and J.Z. performed the experiments. S.L., Y.I., F.G., C.B., A.K., C.M., M.R., and G.L. performed the genotyping and genetic analysis. R.K. and Y.N. provided the HUDEP-2 cell line. M.C.C., S.L., Y.I., L.P., G.-C.Y., and G.L. performed computational and statistical analysis. D.E.B. and S.H.O. supervised this work. M.C.C., D.E.B., and S.H.O. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Daniel E Bauer or Stuart H Orkin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–23 (PDF 23705 kb)

Supplementary Table 1

HbF-associated SNPs. Genome-wide significant SNPs from HbF meta-analysis. (XLSX 10 kb)

Supplementary Table 2

Previously published red blood cell trait associated SNPs22. (XLSX 29 kb)

Supplementary Table 3

Conditional analysis of HbF-associated SNPs. (XLSX 8 kb)

Supplementary Table 4

Genomic cleavage distribution for 8 PAM sequences by chromosome. (XLSX 11 kb)

Supplementary Table 5

Genomic cleavage distribution. Distances between adjacent genomic cleavages for 8 PAM sequences in (a) DHS, (b) enhancers, and (c) repressed regions for 9 ENCODE cell lines as well as (d) RefSeq gene annotations. (XLSX 13 kb)

Supplementary Table 6

NGG-restricted sgRNA library. (XLSX 392 kb)

Supplementary Table 7

NGA-restricted sgRNA library. (XLSX 546 kb)

Supplementary Table 8

sgRNA for Cas9 activity reporters. (XLSX 8 kb)

Supplementary Table 9

MYB shRNA sequences. (XLSX 8 kb)

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Canver, M., Lessard, S., Pinello, L. et al. Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nat Genet 49, 625–634 (2017). https://doi.org/10.1038/ng.3793

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