CRISPR-directed mitotic recombination enables genetic mapping without crosses

Abstract

Linkage and association studies have mapped thousands of genomic regions that contribute to phenotypic variation, but narrowing these regions to the underlying causal genes and variants has proven much more challenging. Resolution of genetic mapping is limited by the recombination rate. We developed a method that uses CRISPR to build mapping panels with targeted recombination events. We tested the method by generating a panel with recombination events spaced along a yeast chromosome arm, mapping trait variation, and then targeting a high density of recombination events to the region of interest. Using this approach, we fine-mapped manganese sensitivity to a single polymorphism in the transporter Pmr1. Targeting recombination events to regions of interest allows us to rapidly and systematically identify causal variants underlying trait differences.. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/040428 doi: bioRxiv preprint first posted online Identification of DNA sequence differences that underlie trait variation is a central goal of modern genetic research. The primary tools for connecting genotype and phenotype are linkage and association studies. In these studies, co-inheritance of genetic markers with the trait of interest in large panels of individuals is used to localize variants that influence the trait to specific regions of the genome. The localization relies on meiotic recombination events that break up linkage between markers on a chromosome. Therefore, the spatial resolution of genetic mapping is limited by the recombination rate.In practice, the recombination rate in most settings is too low to resolve mapped regions to individual genes, much less to specific variants within genes. Increasing mapping resolution requires construction of ever-larger panels of individuals and/or additional generations of recombination, and these approaches are laborious to the point of often being impractical. As a consequence, the genes and variants underlying trait variation remain unidentified for the vast majority of regions implicated by linkage or association mapping.To address this problem, we have devised a new method for genetic mapping that precisely targets recombination events to regions of interest. The method uses recombination events that occur during mitosis rather than meiosis. Rare mitotic recombination events occur naturally when a chromosomal double strand break (DSB) is repaired by homologous recombination (HR) that leads to the formation of a recombined chromosome (Yin and Petes 2013). In a heterozygous individual, cell division can then generate daughter cells with a new genotype that is completely homozygous from the . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/040428 doi: bioRxiv preprint first posted onli...

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