A method for detection of recurrent and non-specific double-stranded DNA break locations in the genome
Inventors: Richard Frock, Jiazhi Hu, Robin Meyers, Frederick Alt
Invention Types: Research Tool
Research Areas: Immunology
Keywords: Assay, Molecular biologyFor More Information Contact: Ives, Catherine L.
Chromosomal translocation occurs when a segment of a chromosome is joined with a nonhomologous chromosome or a site on the same chromosome. This genomic rearrangement frequently results from end-joining of two double-stranded DNA breaks (DSBs). These breaks and subsequent translocations are of great interest in the fields of cancer biology, immunology (VDJ recombination), and molecular biology (gene therapy – CRISPR/Cas, TALENs, ZFNs).
Researchers in Dr. Frederick Alt’s Laboratory at Boston Children’s have developed an improved method for high-throughput genome-wide translocation sequencing (HTGTS) to measure DSB activities due to outside agents or innate biological processes. Their method offers increased sensitivity in identifying off-target hotspots and the occurrence of non-specific DSBs due to the activities of outside agents, including popular gene editing tools such as Cas9 guide RNA endonucleases and TALENs. HTGTS will allow for better characterization of collateral chromosomal damage due to engineered nucleases: a key factor in improving their role in clinical and research applications.
• Engineered nuclease-based HTGTS tool for the study of DNA DSB repair pathways, the mechanisms underlying recurrent genomic alterations, and the processing or re-joining of DSBs
• It is a robust assay for identifying recurrent and non-specific DSB activity
• Universal kit to assess genome-wide DSB activities of various outside agents including engineered nucleases, chemotherapeutics, ionizing radiation, etc.
• Approach allows for improved engineered nuclease design through optimization of specific and non-specific DSB-inducing activities via titration of engineered nuclease levels
• HTGTS method can serve as an assay for off-target and non-specific DSB-inducing activities of engineered nucleases
• New HTGTS approach is inexpensive, fast and robust
• Hu J, Meyers RM, Dong J, Panchakshari RA, Alt FW, Frock RL, 2016. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. PNAS. PMID: 26873106
• Schwer B, Wei PC, Chang AN, Kao J, Du Z, Meyers RM, Alt FW, 2016. Transcription-associated processes cause DNA double-strand breaks and translocation in neural stem/progenitor cells. Nat Protoc. PMID: 27031497
• Frock RL, Hu J, Meyers RM, Ho Y-J, Kii E, Alt FW, 2014. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nature Biotechnology. PMID: 25503383
• Chiarle et al., 2011. Genome-wide Translocation Sequencing Reveals Mechanisms of Chromosome Breaks and Rearrangements in B Cells. Cell 147(1): 107-119. PMID: 21962511
Related Publications: Pat. US 9,518,293 B2