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

Matthew C. Canver, Samuel Lessard, Luca Pinello, Yuxuan Wu, Yann Ilboudo, Emily N. Stern, Austen J. Needleman, Frédéric Galactéros, Carlo Brugnara, Abdullah Kutlar, Colin McKenzie, Marvin Reid, Diane D. Chen, Partha Pratim Das, Mitchel A Cole, Jing Zeng, Ryo Kurita, Yukio Nakamura, Guo Cheng Yuan, Guillaume Lettre & 2 others Daniel E. Bauer, Stuart H. Orkin

Research output: Contribution to journalArticleResearchpeer-review

42 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)625-634
Number of pages10
JournalNature Genetics
Volume49
Issue number4
DOIs
Publication statusPublished - Apr 2017
Externally publishedYes

Keywords

  • computational biology and bioinformatics
  • gene regulation
  • mutagenesis

Cite this

Canver, M. C., Lessard, S., Pinello, L., Wu, Y., Ilboudo, Y., Stern, E. N., ... Orkin, S. H. (2017). Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nature Genetics, 49(4), 625-634. https://doi.org/10.1038/ng.3793
Canver, Matthew C. ; Lessard, Samuel ; Pinello, Luca ; Wu, Yuxuan ; Ilboudo, Yann ; Stern, Emily N. ; Needleman, Austen J. ; Galactéros, Frédéric ; Brugnara, Carlo ; Kutlar, Abdullah ; McKenzie, Colin ; Reid, Marvin ; Chen, Diane D. ; Das, Partha Pratim ; A Cole, Mitchel ; Zeng, Jing ; Kurita, Ryo ; Nakamura, Yukio ; Yuan, Guo Cheng ; Lettre, Guillaume ; Bauer, Daniel E. ; Orkin, Stuart H. / Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. In: Nature Genetics. 2017 ; Vol. 49, No. 4. pp. 625-634.
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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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Canver, MC, Lessard, S, Pinello, L, Wu, Y, Ilboudo, Y, Stern, EN, Needleman, AJ, Galactéros, F, Brugnara, C, Kutlar, A, McKenzie, C, Reid, M, Chen, DD, Das, PP, A Cole, M, Zeng, J, Kurita, R, Nakamura, Y, Yuan, GC, Lettre, G, Bauer, DE & Orkin, SH 2017, 'Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci', Nature Genetics, vol. 49, no. 4, pp. 625-634. https://doi.org/10.1038/ng.3793

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. / Canver, Matthew C.; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily N.; Needleman, Austen J.; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin ; Reid, Marvin; Chen, Diane D.; Das, Partha Pratim; A Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo Cheng; Lettre, Guillaume; Bauer, Daniel E.; Orkin, Stuart H.

In: Nature Genetics, Vol. 49, No. 4, 04.2017, p. 625-634.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Canver, Matthew C.

AU - Lessard, Samuel

AU - Pinello, Luca

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AU - Ilboudo, Yann

AU - Stern, Emily N.

AU - Needleman, Austen J.

AU - Galactéros, Frédéric

AU - Brugnara, Carlo

AU - Kutlar, Abdullah

AU - McKenzie, Colin

AU - Reid, Marvin

AU - Chen, Diane D.

AU - Das, Partha Pratim

AU - A Cole, Mitchel

AU - Zeng, Jing

AU - Kurita, Ryo

AU - Nakamura, Yukio

AU - Yuan, Guo Cheng

AU - Lettre, Guillaume

AU - Bauer, Daniel E.

AU - Orkin, Stuart H.

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