NEW YORK (GenomeWeb) – Measuring the functional effects of genomic variants in a high-throughput manner has been a challenge for researchers. But in a new study published today in Nature Genetics, a team from the University of California, Los Angeles detailed its efforts to develop a CRISPR-library-based approach for highly efficient and precise genome-wide variant engineering that can be used to profile the effects of large classes of variants in a high-throughput way.
The researchers used their method to examine the functional consequences of premature-termination codons (PTCs) at different locations within all annotated essential genes in yeast. They found that most PTCs were highly deleterious unless they occurred close to the 3' end of the gene and did not affect an annotated protein domain. Unexpectedly, they also found that some genes that were previously thought to be essential were actually dispensable, and that other genes had large dispensable regions.
CRISPR-Cas9 editing through homology directed repair (HDR) requires incorporating the desired sequence variants encoded in a DNA template into the genomic locus, and then making sure each cell receives the correct guide RNA-repair template pair. The researchers accomplished this pairing by encoding gRNA targeting sequences and their corresponding repair templates in cis on oligonucleotides generated in bulk through high-throughput synthesis. They then used these oligonucleotide libraries to generate pools of plasmids pairing the two components for delivery into yeast cells.
"We used this approach to understand the consequences of one important class of genetic variants, PTCs. PTCs interrupt the open reading frames of protein-coding genes. Such mutations are generally expected to have strong deleterious effects, either by abrogating or by changing the functions of the encoded proteins or by causing mRNA degradation through the nonsense-mediated decay (NMD) surveillance pathway," the authors wrote. "More than 10 percent of annotated pathogenic human variants are PTCs. Nonetheless, understanding of the detrimental effects of PTCs is incomplete, particularly when they occur near the 3' ends of genes. Such mutations may not shorten the encoded proteins sufficiently to affect their function, and they often escape NMD."
The researchers targeted eight specific PTCs to the Saccharomyces cerevisiae genome and scaled up the approach by using large-scale oligonucleotide synthesis to generate a pool of more than 10,000 distinct paired gRNA-repair template plasmids. These plasmids targeted PTCs to different sites in 1,034 yeast genes considered essential for viability. Each gene was targeted at 10 sites, chosen with a preference for sites closer to the 3' end.
The researchers expected to see PTCs that disrupt the function of genes essential for viability to drop out of the pool over time. They determined the abundance of each barcoded edit-directing plasmid at each time point through bulk sequencing, then computed a PTC tolerance score based on the persistence of the barcoded plasmids over the duration of the experiment.
They found that although most PTCs in annotated essential genes were highly deleterious, some appeared to be tolerated, and that PTCs were generally deleterious when they were located more than 27 codons away from the gene end. Within the 27 terminal codons, the tolerance scores rose toward the 3' end. PTCs were also more tolerated if they did not interrupt or remove an annotated protein domain. PTCs that disrupted protein domains tended to be deleterious even when they were located close to gene ends.
"We computed the overall tolerance of PTCs for each gene and observed considerable variation among genes," the authors wrote. "A Gene Ontology enrichment analysis showed that genes encoding proteins with catalytic activity were significantly less PTC tolerant than other genes, whereas genes with functions relating to mRNA splicing and processing were significantly more PTC tolerant."
They then examined the 16 most PTC-tolerant genes and found that three of those genes had been misannotated as essential because their deletion disrupts the function of a nearby essential gene. This finding, the team noted, illustrated the value of introducing PTCs in order to characterize the essentiality of a gene.
"Our results improve the annotation of essential genes in the well-studied yeast genome. We discovered several cases of genes that appeared to be essential as a consequence of the specific strain and growth conditions originally used to test the viability of gene deletions," the team noted. "These results were consistent with recently reported results based on transposon mutagenesis. A deletion screen in a different yeast isolate has also highlighted examples of conditionally essential genes. Applying our approach and related methods in a diverse set of isolates and growth conditions should further refine the core set of essential yeast genes."
The researchers further added that PTCs are prioritized in studies of human genetic variants because of their high likelihood of abolishing gene function, and that their findings suggest that PTCs are most likely to be deleterious when they disrupt annotated protein domains or truncate more than 27 amino acids, which may improve the filtering of candidate causal variants.