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The resolution of genetic variant mapping is limited by the frequency of recombination events that occur during meiosis and disrupt linkage between adjacent markers. In a study published in Science, Sadhu et al. have circumvented this limitation in yeast by using the CRISPR–Cas system to precisely target recombination events during mitosis, thus enabling higher-resolution mapping of phenotypic traits without the need for meiotic recombination.

The Cas9 endonuclease of the CRISPR system introduces a double-stranded break (DSB) at a target site that is determined by the sequence of a bound guide RNA (gRNA). After the repair of DSBs through homologous recombination, cell division can give rise to daughter cells that exhibit loss of heterozygosity (LOH) between the recombination site and the telomere. Based on this principle, the authors designed 95 gRNAs to target Cas9 to heterozygous sites along an entire chromosome arm in a diploid yeast strain that had been generated by crossing manganese (Mn)-resistant and Mn-sensitive parent strains. They then used fluorescence-activated cell sorting (FACS) to isolate cells that had undergone a LOH event resulting in the loss of a fluoresence gene (encoding GFP), and selected 384 lines representing the 95 different target sites. Whole-genome sequencing identified LOH events in 95% of lines corresponding to sites all along the chromosome arm, and there were few off-target effects, confirming the effectiveness and precision of the CRISPR-assisted recombination.

Next, the group used their LOH panel to map quantitative traits related to growth under 12 different conditions, for which they had previously mapped quantitative trait loci (QTLs) to the chromosome arm under study. Growth on 10 mM Mn sulfate plates mapped to a 2.9 kb interval within a large-effect QTL, whereas eight other growth traits mapped to QTLs with smaller effect sizes. To enable mapping at higher resolution, an additional panel of LOH lines was generated using three gRNAs directed at sites near the interval associated with Mn sensitivity. Sequencing revealed that 13.1% of resultant LOH cell lines had a recombination event within the 2.9 kb interval; importantly, almost all of the variants within the interval were separated by recombination events in the panel. By contrast, only 0.7% of cells whose recombination events were generated through random meiotic segregation had recombination within this interval. These figures indicate that 7,500 strains generated by conventional crossing would be required to obtain a number of recombination events comparable to that obtained by CRISPR-targeted recombination.

Further growth experiments were carried out to assess the Mn sensitivity of the fine-mapping panel. By comparing the observed phenotypes with the locations of recombination breakpoints, the authors were able to pinpoint Mn resistance to a single polymorphism present in one of the parent yeast strains. Introduction of this variant, but not neighbouring variants, resulted in Mn resistance in the previously sensitive parent strain, demonstrating the accuracy of the CRISPR-assisted fine-mapping approach.

The high density of recombination events produced using this technique (which negates the need for multiple generations of crossing) combined with its targetability, holds promise for the further refinement of trait mapping in unicellular organisms. Furthermore, the authors suggest that this technique could also be applied to cultured human cells to facilitate the elucidation of traits that have measurable cellular phenotypes.