BLESS

BLESS

BLESS is a genome-wide approach to map DSBs at nucleotide resolution. 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.

Pros:

• Detects DSBs at nucleotide resolution
• Does not depend on proteins that bind to DSBs—a source of bias
• Does not depend on single-stranded DNA (ssDNA)—a source of bias

Cons:

• High background; only maps unjoined ends¹
• Susceptible to artifacts associated with cell fixation²

• BLESS: Crosetto N., Mitra A., Silva M. J., Bienko M., Dojer N., et al. (2013) Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods 10: 361-365
• 1. Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W., et al. (2016) Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc 11: 853-871
• 2. Tsai S. Q., Zheng Z., Nguyen N. T., Liebers M., Topkar V. V., et al. (2015) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33: 187-197

1. Ran F. A., Cong L., Yan W. X., Scott D. A., Gootenberg J. S., et al. (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520: 186-191