A CRISPR-Cas9-based tool for dose-dependent DNA damage detection

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions that trigger complex cellular responses.
• Understanding the DNA damage response (DDR) is crucial for fields ranging from cancer research to aging.
• Existing methods for inducing DSBs often lack precise control over break frequency and location.

Purpose of the Study:

• To develop a novel CRISPR-Cas9-based system for precise and dose-dependent induction of DSBs in Saccharomyces cerevisiae.
• To investigate the dynamics and localization of key DDR proteins, such as Tel1 kinase, following controlled DSB induction.
• To establish a scalable platform for studying genome stability and DDR mechanisms across different organisms.

Main Methods:

• Utilized a CRISPR-Cas9 system targeting Ty retrotransposons in yeast for sequence-specific DSB induction.
• Controlled the number of induced DSBs (×1, ×15, or ×59) to enable dose-dependent studies.
• Employed microscopy techniques to observe the localization and foci formation of the Tel1 kinase in response to DSBs.

Main Results:

• The developed system allowed for precise, dose-dependent induction of DSBs, facilitating detailed DDR studies.
• Tel1 kinase was observed to localize to the nuclear periphery and form multiple foci upon DSB induction.
• The study identified limitations in Cas9 availability at higher break induction levels, providing insights for system optimization.

Conclusions:

• The novel CRISPR-Cas9 system offers a powerful and scalable tool for dissecting DNA damage response pathways.
• Precise control over DSB induction enhances the ability to study DDR dynamics and protein localization.
• This platform has broad applicability for investigating genome stability and DDR mechanisms in various model organisms.