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Updated: Dec 5, 2025
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout
Merve Dede1,2, Megan McLaughlin1,2, Eiru Kim1
1Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Background:
Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell.
Results:
Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes.
Conclusions:
Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.
Insights
Functionally redundant paralogs, often missed in single-gene CRISPR screens, represent a significant, targetable source of cancer dependencies. These gene pairs offer new avenues for cancer therapy development.
Area of Science:
- Cancer Biology
- Genomics
- Synthetic Lethality
Background:
- CRISPR/Cas9 knockout screens identify essential genes for cancer target discovery.
- Monogenic screens detect fewer essential genes than constitutively expressed genes.
Purpose of the Study:
- Investigate why many constitutively expressed genes are not detected in CRISPR screens.
- Identify functionally redundant paralogs as potential cancer dependencies.
Main Methods:
- Systematic analysis of Cancer Dependency Map CRISPR screen data.
- CRISPR/enCas12a dual-gene knockout screening of paralog pairs.
- Functional buffering analysis in three cancer cell lines.
Main Results:
- Half of constitutively expressed genes were never detected in CRISPR screens, enriched for paralogs.
- Identified 24 synthetic lethal paralog pairs missed by monogenic screens.
- 79% of synthetic lethal paralogs were validated across multiple cell lines.
Conclusions:
- Functionally redundant paralogs are a targetable genetic dependency class.
- These paralogs are under-represented in standard monogenic CRISPR screens.
- Dual-gene knockout screening reveals novel cancer dependencies.
