COLD SPRING HARBOR, NY (GenomeWeb) – Researchers have developed a new tool based on the CRISPR/Cas9 gene-editing approach to identify functional genetic differences between species, populations, and individuals.
Genome-wide association studies have generated lists of genetic loci associated with a number of traits and conditions, but it has been difficult for researchers to drill down to causal variants.
"It's hard to detect the cause of phenotypic differences," Eilon Sharon, a postdoctoral researcher at Stanford University, said during his talk at the Biology of Genomes meeting held here this week. The result is, he added, that researchers still don't know the variants that underlie certain traits.
Sharon presented a new high-throughput genome-wide genome editing-based tool he and his colleagues developed to find those functional variants. During his talk, which was during a session devoted to genome engineering and editing, Sharon said that this tool was able to provide single-nucleotide resolution when applied to uncover the fitness consequences of genetic differences between two yeast strains.
The tool, dubbed CRISPeY, is a CRISPR/Cas9 based approach that relies on homology-directed repair. When the CRISPR/Cas9 gene-editing tool makes double-stranded breaks in DNA, it can lead to either non-homologous end joining or homology-directed repair, depending. While both fix the cut, non-homologous end joining can knock out a gene, while homology-directed repair can use a template to seal the cut and introduce that donor DNA. However, Sharon said, homology-directed repair generally has low efficiency.
In CRISPeY, both the guide and donor RNA are included on the same 200 base-pair oligonucleotide which, along with other tweaks, can boost its efficiency, he said. Using this CRISPeY approach, Sharon said researchers could introduce thousands of different, specific genetic variants in one experiment.
In wild-type yeast, he reported that CRISPeY led to near-perfect editing with no evidence of non-homologous end joining. Further, he said the approach had more than 93 percent efficiency.
With CRISPeY, Sharon said they could now conduct genome-wide precise editing studies. He and his colleagues used it to examine the fitness consequences of the 16,006 SNPs and indels that differ between the laboratory yeast strain BY and the vineyard yeast strain RM.
They transfected, edited, and grew up the two yeast lines on glucose media and examined their fitness every two to three generations. Overall, they uncovered 572 variants with significant differences in fitness. In particular, Sharon reported that 171 of these variants had a greater than 1 percent effect on fitness.
He noted, though, that the study was better able to detect positive effects on fitness.
These significant variants with large effects, he added, were enriched within regulatory regions, especially in promoter regions. In particular, they found functional variants to be enriched in and near transcription factor binding sites. Only about 19 percent of these significant variants with large effects influence protein-coding regions, the researchers noted in their abstract.
When they searched for signals of selection, Sharon said he and his colleagues found that ribosomal gene promoters are enriched for fitter RM alleles. This indicated to them that there has been polygenic adaptation of the ribosome.
Sharon and his colleagues also noticed that many of the strong-effect alleles also appeared to cluster together. These nearby variants nearly always came from the same parent, which also indicated to them that lineage-specific selection on genes could be driven by multiple variants.
Going forward, Sharon said he would also like to apply the approach to study humans.