A Whole Genome CRISPR/Cas9 Screening Approach for Identifying Genes Encoding DNA End-Processing Proteins

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions repaired by homologous recombination (HR) and non-homologous end joining (NHEJ).
• The choice between HR and NHEJ pathways depends on DNA end resection, generating single-stranded DNA (ssDNA) overhangs bound by Replication Protein A (RPA).
• Understanding the regulation of DNA end processing is vital for comprehending genome stability and DNA repair mechanisms.

Purpose of the Study:

• To develop and present novel protocols for studying DNA end processing pathways.
• To identify and characterize new regulators involved in the choice between HR and NHEJ.
• To establish a system for screening genetic modifiers of DNA repair using CRISPR/Cas9 technology.

Main Methods:

• Generation of Abelson murine leukemia virus-transformed pre-B cells (abl pre-B cells) with stably integrated inducible Cas9.
• CRISPR/Cas9-mediated gene inactivation for functional genomics screens.
• Whole genome guide RNA (gRNA) library screening coupled with flow cytometry for RPA chromatin binding detection.

Main Results:

• Successful establishment of a versatile cell system for DNA repair pathway investigation.
• Identification of novel genes and pathways influencing DNA end processing and RPA loading.
• Demonstration of flow cytometry as an effective method for detecting RPA engagement after DNA damage.

Conclusions:

• The developed protocols provide a powerful platform for discovering novel regulators of DNA double-strand break repair.
• These findings advance our understanding of the intricate mechanisms governing DNA end processing.
• The study highlights the importance of RPA binding in directing DNA repair pathway choice.