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CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates.
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Are there increases in CRISPR/Cas off-target effects in homologous recombination repair deficient Saccharomyces cerevisiae cells?
CRISPR/Cas technology is increasingly being used as a common methodology in many cancer biology studies due to the ease and convenience of the technique. Precise editing of genomic DNA has been achieved upon repair of CRISPR-induced DNA double-strand breaks (DSBs) by homologous recombination (HR). HR repairs DNA DSBs with high fidelity and therefore, deficiencies in HR result in genome instability. These deficiencies have been demonstrated in many cancers. RAD51-dependent HR is a very important pathway for repairing DSBs. Previous studies have shown that genome editing using CRISPR technology relies on the repair of site-specific DNA DSBs induced by the RNA-guided Cas9 endonuclease. Furthermore, previous studies have shown that the efficiency of CRISPR-mediated HR can be improved by the stimulation of HR promoting factors, such as the RAD51 recombinase. Despite the ease and efficient use the CRISPR/Cas technology for genome editing, one limitation is the potential occurrence of associated off-target effects. If CRISPR technology is planned to be used to target cancer cells with defective HR capabilities, will off-target mutations be likely to occur? In order to answer this question, a system was developed in Saccharomyces cerevisiae using green fluorescent protein (GFP) as a reporter to identify off-target CRISPR-induced DSBs. This study set out to test the number of off-target DSBs that could be introduced by CRISPR-induced genome editing in a RAD51-deficient HR model. We were curious whether loss of RAD51-dependent HR would increase the abundance of off-target CRISPR-induced DSBs in mutant yeast strains as compared to those with a functioning HR-dependent DNA repair pathway. Preliminary findings using this system will be presented.
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- Award ID(s):
- 2027813
- PAR ID:
- 10464942
- Date Published:
- Journal Name:
- Southeastern biology
- Volume:
- 80
- Issue:
- 1-4
- ISSN:
- 1533-8436
- Page Range / eLocation ID:
- 93
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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