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Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens
Published on: June 6, 2020
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Optimizing gRNA selection for high-penetrance F0 CRISPR screening for interrogating disease gene function.
Sheng-Jia Lin1, Kevin Huang1, Cassidy Petree1
1Genes & Human Disease Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, United States.
Nucleic Acids Research
|March 19, 2025
Summary
This study introduces new guide RNA selection rules for CRISPR/Cas9 genome editing in zebrafish. This method enables high-throughput screening of gene functions and accelerates the discovery of genes linked to human diseases.
Area of Science:
- Genetics and Genomics
- Developmental Biology
- Molecular Biology
Background:
- Identifying causative genes for human diseases is challenging.
- CRISPR/Cas9 genome editing in zebrafish (F0 Crispants) aids in gene function validation but faces limitations in high-throughput screening due to variability and low penetrance.
- Existing methods are costly and inefficient for large-scale genetic screens.
Purpose of the Study:
- To develop optimized guide RNA (gRNA) selection rules for high phenotypic penetrance in F0 zebrafish knockouts.
- To establish a robust, high-throughput pipeline for rapid characterization of candidate human disease genes.
- To enable efficient genetic screening for novel disease-associated genes.
Main Methods:
- Developed specific guide RNA (gRNA) selection rules for CRISPR/Cas9 genome editing.
- Achieved high phenotypic penetrance with 1-2 gRNAs per gene, enabling up to three simultaneous knockouts in F0 zebrafish.
- Validated the approach across 324 gRNAs targeting 125 genes, assessing transcriptomic overlap with stable knockout lines.
- Applied the method for high-throughput screening of neurodevelopmental and hearing genes.
Main Results:
- Demonstrated high phenotypic penetrance (up to three simultaneous knockouts) in F0 zebrafish using optimized gRNA selection.
- Showcased strong transcriptomic overlap between F0 knockouts and stable knockout lines.
- Successfully identified 10 novel neurodevelopmental disorder genes and 50 hearing genes through high-throughput screening.
- Utilized the approach to study gene epistasis and characterize paralogous genes.
Conclusions:
- Optimized gRNA selection rules significantly enhance phenotypic penetrance in F0 zebrafish CRISPR/Cas9 knockouts.
- This approach provides a rapid, cost-effective, and robust pipeline for validating candidate human disease genes.
- The method facilitates high-throughput genetic screening, accelerating the discovery of novel genes involved in human diseases.
