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

Pubmed ID: 28218758

Pubmed Central ID: PMC5374001

Journal: Nature genetics

Publication Date: April 1, 2017

Affiliation: Howard Hughes Medical Institute, Boston, Massachusetts, USA.

MeSH Terms: Humans, Genome-Wide Association Study, Cells, Cultured, Mutagenesis, Genetic Variation, Gene Dosage, Quantitative Trait Loci, DNA, Intergenic, HEK293 Cells, Regulatory Elements, Transcriptional

Grants: R01 HL119099, K08 DK093705, R01 HL032259, R03 DK109232, DP2 HL137300, P30 DK049216, K99 HG008399, P01 HL032262, R00 HG008399, U54 DK106829, F30 DK103359

Authors: Brugnara C, Lettre G, Orkin SH, Nakamura Y, Wu Y, Kutlar A, Canver MC, Lessard S, Pinello L, Ilboudo Y, Stern EN, Needleman AJ, Galactéros F, McKenzie C, Reid M, Chen DD, Das PP, A Cole M, Zeng J, Kurita R, Yuan GC, Bauer DE

Cite As: 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. Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nat Genet 2017 Apr;49(4):625-634. Epub 2017 Feb 20.

Studies:

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.