CRISPR-StAR enables high-resolution genetic screening in complex in vivo models

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Pooled CRISPR-Cas9 screening enables genome-wide gene-phenotype mapping.
• Assessing genetic perturbations requires large cell numbers to mitigate stochastic drift.
• Complex models like organoids and mouse xenografts present scaling challenges due to heterogeneity and bottlenecks.

Purpose of the Study:

• To introduce CRISPR-StAR, a novel screening method overcoming limitations of conventional pooled genetic screens.
• To generate clonal, single-cell-derived intrinsic controls for enhanced data quality.
• To identify in vivo-specific genetic dependencies in complex biological systems.

Main Methods:

• CRISPR-StAR activates single-guide RNAs (sgRNAs) in half of each cell's progeny post-re-expansion.
• This generates intrinsic controls within clonal cell populations.
• The method was applied to a genome-wide screen in mouse melanoma models.

Main Results:

• CRISPR-StAR effectively overcomes intrinsic and extrinsic cellular heterogeneity.
• The method mitigates genetic drift encountered in bottleneck events.
• Benchmarking demonstrated superior data quality compared to conventional screening approaches.

Conclusions:

• CRISPR-StAR provides a robust solution for high-resolution genetic screening in complex and heterogeneous models.
• The technology enables more accurate identification of gene functions and dependencies.
• This advancement facilitates the study of in vivo-specific genetic dependencies, particularly in cancer research.