The Promise of Paired Knockouts in Oncology
Introduction
Most CRISPR screens today focus on knocking out one gene at a time. That approach has fueled remarkable discoveries, but it leaves blind spots. Many genes in our genome have “backups”, genes that compensate when one is lost. For cancer researchers, this means that critical vulnerabilities can remain hidden unless you look at gene pairs together.
Redundancy in Cancer
Cells are remarkably resilient because many genes have overlapping or compensatory functions. Paralogs, genes that arise through duplication events, are one common source of this redundancy, but they are not the only ones. Sometimes functionally similar but unrelated genes can step in for each other as well. In healthy cells, this redundancy provides stability, but in cancer, it can mask therapeutic opportunities. When one gene in a redundant pair is already mutated or deleted, the partner can become essential for tumor survival. This is a classic setup for synthetic lethality, where inactivating both genes proves lethal, even though either one alone is dispensable. A recent review emphasized that paralog targeting represents one of the most promising but underexplored areas of synthetic lethality in oncology.
Synthetic Lethality in Cancer
The most famous example of synthetic lethality is the use of PARP inhibitors in tumors with BRCA1/2 mutations. By blocking DNA repair through both pathways at once, cancer cells collapse under the pressure, while healthy cells remain intact. Researchers believe that paralog pairs across the genome harbor many more such opportunities, but they remain largely untapped.
Enter Combinatorial CRISPR Screens
Combinatorial CRISPR technology makes it possible to knock out two genes simultaneously. Rather than testing every possible pair, an impossible task given the combinatorial explosion, researchers focus on targeted sets of genes. The goal: uncover gene dependencies that only emerge in pairs. A Cas12a-based “in4mer” platform recently scaled this up by targeting ~4,000 paralog pairs, showing how technology is catching up to the ambition of combinatorial screening.
In oncology, these paired screens could reveal entirely new drug targets. If a tumor already carries a loss-of-function mutation in one gene, targeting its functionally-redundant counter-part could offer a selective and potent therapeutic strategy. Indeed, a recent pan-cancer study testing 472 gene pairs identified 117 robust synthetic lethal interactions, underscoring just how powerful this approach can be.
The Analysis Bottleneck
Analyzing paired knockout screens is not straightforward. Most bioinformatics tools were designed for single-gene knockouts, and they fall short when applied to more complex data. Quality control is more difficult, compensation mechanisms are layered, and interpretation requires new statistical frameworks. Benchmarking efforts have shown that normalized Z-scores outperform traditional approaches for calling hits in multiplex screens, emphasizing the importance of computational rigor in this space..
Data: Open and Closed Doors
The Cancer Dependency Map (DepMap) has made single-gene knockout data freely available, democratizing access to large-scale functional genomics. But paired-knockout datasets remain limited and often locked behind paywalls. Facilities like the Broad Institute provide sequencing services for combinatorial screens, and dual-guide CRISPRi studies have already begun to systematically map synthetic lethal interactions in critical pathways such as the DNA damage response.
Why This Matters
The promise of combinatorial CRISPR is clear: uncovering synthetic lethal gene pairs could dramatically expand the set of druggable targets in cancer. By focusing on the dependencies that arise only when redundancy is stripped away, researchers may find new ways to selectively target tumors while sparing healthy tissue.
Looking Ahead
As technology matures, expect rapid growth in both experimental methods and computational frameworks for combinatorial CRISPR. For bioinformatics teams, the challenge is to design tools that can handle the combinatorial explosion, extract meaningful insights, and integrate with existing resources like DepMap.
At Bridge Informatics, we see this as a frontier where wet-lab discovery and computational analysis must advance hand in hand. Knocking out one gene at a time is no longer enough. The future lies in pairs and the hidden vulnerabilities they reveal.
Ready to unlock the full power of combinatorial CRISPR?Click here to schedule a call with BI to discuss how we can help you analyze complex paired knockout datasets, build scalable QC and hit-calling pipelines, and accelerate your synthetic lethal discovery efforts.