Open AccessA Model to Investigate Single-Strand DNA Responses in G1 Human Cells via a Telomere-Targeted, Nuclease-Deficient CRISPR-Cas9 System
Author(s) -
Remco Paul Crefcoeur,
Omar Zgheib,
Thanos D. Halazonetis
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0169126
Subject(s) - telomere , biology , cas9 , crispr , dna replication , replication protein a , dna , dna repair , microbiology and biotechnology , rad51 , nuclease , control of chromosome duplication , genetics , gene , dna binding protein , transcription factor
DNA replication stress has the potential to compromise genomic stability and, therefore, cells have developed elaborate mechanisms to detect and resolve problems that may arise during DNA replication. The presence of single-stranded DNA (ssDNA) is often associated with DNA replication stress and serves as a signal for both checkpoint and repair responses. In this study, we exploited a CRISPR-Cas9 system to induce regions of ssDNA in the genome. Specifically, single-guide RNAs bearing sequence complementarity to human telomeric repeats, were used to target nuclease-deficient Cas9 (dCas9) to telomeres. Such targeting was associated with the formation of DNA-RNA hybrids, leaving one telomeric DNA strand single-stranded. This ssDNA then recruited DNA repair and checkpoint proteins, such as RPA, ATRIP, BLM and Rad51, at the telomeres. Interestingly, targeting of all these proteins to telomeric ssDNA was observed even in cells that were in the G1 phase of the cell cycle. Therefore, this system has the potential to serve as a platform for further investigation of DNA replication stress responses at specific loci in the human genome and in all phases of the cell cycle.