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

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

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Title: Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci
Author: 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; Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel E.; Orkin, Stuart H.

Note: Order does not necessarily reflect citation order of authors.

Citation: Canver, M. C., S. Lessard, L. Pinello, Y. Wu, Y. Ilboudo, E. N. Stern, A. J. Needleman, et al. 2017. “Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.” Nature genetics 49 (4): 625-634. doi:10.1038/ng.3793. http://dx.doi.org/10.1038/ng.3793.
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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 from genome-wide association studies largely cluster in regulatory DNA. 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 combinatorial nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, a locus 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, which emphasizes the importance of off-target analysis in design of saturating mutagenesis experiments. Taken together, these data establish a widely applicable high-throughput and high-resolution methodology to reliably identify minimal functional sequences within large regions of disease- and trait-associated DNA.
Published Version: doi:10.1038/ng.3793
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374001/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:34375299
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