BLESS
Breaks labeling and Enrichment on Streptavidin and Sequencing
BLESS is a genome-wide approach to map DSBs at nucleotide resolution (Crosetto et al., 2013). BLESS is able to detect telomere ends, Sce endonuclease_induced DSBs, and complex genome-wide DSB landscapes.In this method, DSBs are ligated in situ to a proximal linker covalently linked to biotin. The gDNA is extracted and fragmented, and the labeled fragments are captured on streptavidin beads. Next, a distal linker is ligated to the free end of the captured fragments. The fragments are released by linker digestion with I-SceI and PCR-amplified
Advantages:
- Detects DSBs at nucleotide resolution
- Does not depend on proteins that bind to DSBsãa source of bias
- Does not depend on single-stranded DNA (ssDNA)ãa source of bias
Disadvantages:
- High background; only maps unjoined ends (Hu et al., 2016)
- Susceptible to artifacts associated with cell fixation (Tsai et al., 2015)
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Lee C. M., Cradick T. J., Fine E. J. and Bao G. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing. Mol Ther. 2016;24:475-487
References:
Ran F. A., Cong L., Yan W. X., et al. in vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520:186-191