An important consideration in gene editing technology.
Typically checked through several complementary approaches, including:
- Computational Prediction: computational tools to predict potential off-target sites based on sequence similarity to the target site
- Tools like Cas-OFFinder or COSMID scan the genome for sequences that match the guide RNA with some allowed mismatches
- These generate a list of potential off-target sites to check experimentally
- Experimental Validation:
a) Targeted Sequencing:
- PCR amplification of predicted off-target sites
- Deep sequencing of these regions to detect any indels (indel @ off-target site = evidence of off-target editing)
- GUIDE-seq: Uses integration of double-stranded oligonucleotides at DNA break sites
- DISCOVER-seq: Tracks DNA repair factors to identify where Cas9 cuts
- CIRCLE-seq: Uses cell-free DNA to identify potential cut sites
- These methods can find unpredicted off-target sites
- Analysis:
- Indel frequencies are calculated at each site (see example data here: In Vivo Genome Editing by Lipid Nanoparticle Delivery of a Stable CRISPR-Cas9 Ribonucleoprotein - Kai Chen Seminar) b) Unbiased Methods:
- Usually presented as a percentage of total reads showing mutations
- Sites with indel rates significantly above background are considered off-target effects
For therapeutic applications, even more stringent methods might be used, including whole genome sequencing to look for any unexpected mutations.