NEW YORK (GenomeWeb) – A team led by researchers at the University of California, San Francisco used CRISPR interference (CRISPRi) to map genetic interactions in human cells at a large scale.
As they wrote yesterday in Cell, yeast studies have shown that comprehensively mapping genetic interactions (GIs) can help investigators infer gene function. However, until this point, it has not been feasible to generate large-scale, diverse human GI maps.
For this study, the researchers developed a CRISPRi platform to systematically perturb 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, and assign function to poorly characterized genes, including TMEM261, a new electron transport chain component.
"Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway," the authors wrote. "Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells."
The team used CRISPRi to systematically inactivate pairs of genes in an acute lymphoblastic leukemia cell line and in a chronic myeloid leukemia cell line. This resulted in a map of 111,628 unique two-gene interactions, and the researchers then clustered the 472 genes according to their relationships with one another. They were also able to assign these clusters to specific biological pathways or locations within the cell.
The new gene interaction maps captured 80 percent of known functional relationships between the genes being studied, the researchers noted, but they also found that the majority of strong interactions revealed by the new data were novel, including many gene pairs which were not known to interact directly but had been independently associated with the formation of protein complexes or with cellular processes such as energy production.
Further, the researchers found a novel synthetic lethal relationship between cellular pathways regulating cholesterol metabolism and those regulating DNA damage repair. Specifically, they observed that when they inactivated FDPS, which is involved in producing cholesterol, cells became highly dependent on the HUS1 DNA repair gene for survival.
The FDPS gene is responsible for modifying a chemical called IPP as it produces cholesterol. When FDPS is suppressed, IPP builds up in the cell and may cause DNA damage that requires repair for the cell to survive. Importantly, FDPS is the target for the bisphosphonate anti-cholesterol drugs, which can also increase bone density. This has made them one of the leading treatments for osteoporosis, though it has not been clear until now why an anti-cholesterol drug affects bone density. This study suggest that bisphosphonates may trigger DNA damage via buildup of IPP in osteoclasts.
"Our work provides a robust platform for future GI mapping efforts that will complement the rich insights obtained from recent large-scale efforts that use comparative genome-scale CRISPR or RNAi screens in the context of naturally occurring cancer-associated genome variations across cancer cell lines to define gene function," the authors wrote. "Beyond the cancer genome, we envision applying CRISPR-based methods to model disease-associated cellular states (genomic variants, transcriptional profiling, and epigenetic profiling) and then using GI maps to dissect specific disease states with high resolution. Although, here, we focus on cell growth, we anticipate that these approaches can be applied to any quantifiable measure of cellular phenotype."