OffTargets

Off-targets refer to unintended molecules, sequences, or sites that interact with a given reagent or tool outside the intended target. In biology and medicine, off-target activity can occur with genome-editing nucleases, RNA interference reagents, antisense oligonucleotides, and small-molecule drugs. Off-target effects may produce unintended genetic changes, gene silencing, or physiological side effects, complicating interpretation of experiments and raising safety concerns for therapies.

In genome editing, off-targets usually arise when a guide RNA directs a nuclease to sequences similar to the intended site. Unintended cleavage or binding can lead to insertions, deletions, chromosomal rearrangements, or cryptic transcription. Off-target events are influenced by sequence similarity, chromatin accessibility, and nuclease properties. Common methods to detect off-targets include GUIDE-seq, DISCOVER-Seq, CIRCLE-seq, and SITE-Seq.

Strategies to reduce off-target activity include designing gRNAs with high specificity, using high-fidelity nucleases (for example, Cas9 variants such as SpCas9-HF1, eSpCas9, HypaCas9), employing truncated or chemically modified guides, and using paired nickases or base editors. Delivery methods that limit exposure and rigorous off-target screening before clinical use are also employed.

In pharmacology and molecular biology, off-target effects are evaluated through in vitro profiling, animal studies, and clinical observation. Understanding and mitigating off-targets is essential for interpreting experimental outcomes and ensuring safety in gene therapies and drug development.