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In-vitro Mutagenesis

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

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Updated: Jun 27, 2025

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Published on: September 4, 2019

Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform.

Nazanin Esmaeili Anvar^1,2, Chenchu Lin¹, Xingdi Ma^1,2

¹Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Nature Communications

|April 27, 2024

Summary

We developed a new Cas12a-based genetic screening platform, in4mer, for scalable analysis of gene interactions in mammalian cells. This method efficiently identifies paralog synthetic lethals, reducing costs and improving assay performance.

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Area of Science:

* Genomics and Molecular Biology
* Mammalian Cell Biology
* Genetic Screening Technologies

Background:

* Understanding genotype-phenotype relationships requires analyzing genetic interactions.
* Current combinatorial genetic perturbation technologies in mammalian cells face scalability challenges.
* Previous CRISPR screens identified paralog synthetic lethals, but sensitivity and reproducibility can be improved.

Purpose of the Study:

* To develop a scalable and sensitive platform for combinatorial genetic perturbation in mammalian cells.
* To identify background-independent paralog synthetic lethals.
* To enhance the efficiency and reduce the cost of genetic interaction assays.

Main Methods:

* Development of the in4mer Cas12a platform utilizing arrays of four independent guide RNAs.
* Construction of the genome-scale Inzolia library targeting approximately 4000 paralog pairs.
* Application of the platform in cancer cells for genetic interaction screening.

Main Results:

* The in4mer Cas12a platform demonstrated superior sensitivity and assay replicability compared to previous methods.
* The Inzolia library, ~30% smaller than typical CRISPR/Cas9 libraries, effectively screened paralog pairs.
* Screens successfully discriminated essential genes and detected synthetic lethal, masking, and buffering interactions between paralogs.
* The in4mer platform achieved a fivefold reduction in library size compared to other genetic interaction methods.

Conclusions:

* The in4mer Cas12a platform offers a scalable, cost-effective, and highly sensitive approach for analyzing genetic interactions in mammalian cells.
* This technology significantly advances the study of paralog synthetic lethality and complex genetic interactions.
* The developed platform has broad implications for functional genomics and disease research.