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Optimizing photoactivation of PA-mCherry for optical pooled CRISPR screens
Sravasti Mukherjee1,2,3, Giulia Zanetti1,3, Bram van den Broek1,3,4
1Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Abstract:
Optical pooled CRISPR screens have become an attractive tool for the rapid identification of genes involved in biological processes. In such screens, mixed populations of cells, each with a single gene knocked out, are screened by microscopy for phenotypes of interest. Identified hit cells can then be tagged by photoactivation of a co-expressed marker, such as PA-mCherry, and subsequently isolated by FACS to identify the responsible guide RNA by next-generation sequencing. Photoactivation is typically performed by selective irradiation of cells with UV light, using either a digital mirror device (DMD), an external fixed UV laser, or, conveniently, by using the 405 nm laser line present in most confocal scanning microscopes. In this study, the latter approach is optimized for PA-mCherry, a bright red phototag used by us and others in optical pooled screens. We find that although normal scanning with intense 405 nm light can rapidly activate PA-mCherry, it also leads to rapid photobleaching. Instead, much higher cellular brightness is achieved by limiting intensity and pixel dwell time during scanning, as well as by slightly defocusing the laser. These results should help optimize cell tagging for genotype-phenotype mapping in optical pooled screens, as well as for other applications.
Insights
Optimizing photoactivation of PA-mCherry in optical pooled CRISPR screens enhances gene discovery. Modified laser settings improve cellular brightness for accurate genotype-phenotype mapping.
Area of Science:
- * Molecular Biology
- * Genetics
- * Cell Biology
Background:
- * Optical pooled CRISPR screens enable rapid gene identification by linking cellular phenotypes to specific gene knockouts.
- * Photoactivatable markers like PA-mCherry are crucial for tagging and isolating cells of interest for downstream genetic analysis.
- * Confocal microscopy's 405 nm laser line offers a convenient method for photoactivation, but requires optimization.
Purpose of the Study:
- * To optimize the photoactivation of PA-mCherry using a confocal microscope's 405 nm laser for improved efficiency in optical pooled screens.
- * To enhance cellular brightness and minimize photobleaching during PA-mCherry activation.
- * To provide guidelines for improved cell tagging in genotype-phenotype mapping applications.
Main Methods:
- * Investigated PA-mCherry photoactivation using a 405 nm laser on a confocal scanning microscope.
- * Systematically varied laser intensity, pixel dwell time, and focal distance during photoactivation.
- * Assessed cellular brightness and photobleaching levels under different activation conditions.
Main Results:
- * Standard high-intensity 405 nm laser scanning rapidly activates PA-mCherry but causes significant photobleaching.
- * Reduced laser intensity and pixel dwell time, along with slight defocusing, significantly increased PA-mCherry cellular brightness.
- * Optimized parameters yielded improved signal for cell tagging and subsequent genetic identification.
Conclusions:
- * Optimized 405 nm laser settings enhance PA-mCherry photoactivation efficiency and signal quality in optical pooled screens.
- * These findings facilitate more effective genotype-phenotype mapping by improving cell tagging and isolation.
- * The optimized protocol benefits broader applications requiring precise photoactivation of PA-mCherry.
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