WP3 will develop and exploit methods to assess genome toxicity at different scales, covering from small lesions to chromosomal scale unintended modifications. Sensitive methodologies to evaluate off-target activities of the editing technologies developed in WP1 and WP2 will be developed. Circle-seq methodology will be used to address programmable nuclease potential offtargets (WP1).
Epigenetic editing off-target profile will be assessed using ChIP-Seq and RNAseq. Insertional mutagenesis of WP2 will be carefully profiled using a methodology based on detection of genome-cassette junctions. Programmable nucleases long range chromosomal rearrangements will be addressed by combining potential off-target probes and nanopore long-read sequencing. Genome engineering consequence are dependent on the specific cell type. Genome toxicity will be assessed from clinically relevant in vitro and in vivo models used in the proposal (WP6-WP9).
Specific WP’s objectives:
- Determining the genome-wide specificity profiles of gene editing nucleases in cultured mammalian cells and in vivo
- Novel assay for defining the genome-wide off-target effects of designer recombinases
- Novel method for profiling genome-wide off-target insertional mutagenesis
- Novel assays for identifying genome toxicity at the chromosomal level
- Determining the genome-wide effects of epigenetic editing tools in mammalian cells
- Task 3.1: Defining the genome-wide specificity profiles of gene editing nucleases in mammalian cells in culture and in vivo
- Task 3.2: A new method to define genome-wide off-target effects of designer recombinases
- Task 3.3: Novel method to profile genome-wide off-target insertions of the donor DNA template
- Task 3.4: Long range chromosomal rearrangements using long read technologies
- Task 3.5. Defining the genome-wide specificity of epigenetic editing tools
WP leader: Keith Joung (MGH)
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