Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq.
Beeke WienertStacia K WymanChristopher D RichardsonCharles D YehPinar AkcakayaMichelle J PorrittMichaela MorlockJonathan T VuKatelynn R KazaneHannah L WatryLuke M JudgeBruce R ConklinMarcello MarescaJacob E CornPublished in: Science (New York, N.Y.) (2019)
CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.
Keyphrases
- crispr cas
- genome editing
- dna damage
- dna repair
- single cell
- induced apoptosis
- small molecule
- rna seq
- genome wide
- high throughput
- induced pluripotent stem cells
- single molecule
- oxidative stress
- endoplasmic reticulum stress
- signaling pathway
- drug delivery
- skeletal muscle
- cell proliferation
- real time pcr
- cancer therapy
- dna damage response
- nucleic acid
- cell death
- wild type
- pi k akt