Comprehensive Off-Target Assessment Strategies For CRISPR-Based Genome Editing Therapeutics: Integrating Biochemical, Bioinformatic, And Unbiased Approaches

Off-target safety evaluation is one of the most consequential challenges in developing CRISPR-based therapeutics, and no single detection method captures the full spectrum of risk. Researchers at ElevateBio present a multi-layered framework that combines Digenome-Seq, homology-based computational prediction, and whole exome sequencing to nominate and confirm off-target editing events across nuclease, adenosine base editor, and cytosine base editor modalities.

Key findings show that off-target profiles vary significantly by cell type and editor engineering, underscoring the importance of evaluation in therapeutically relevant primary cells from both male and female donors. The data also demonstrate that iterative improvements to the adenosine deaminase domain reduce both the number and magnitude of off-target events, and that WES can nominate edits at allele frequencies as low as 1%. Confirmed sites are further contextualized through clinical variant annotation and global genomic and transcriptomic safety evaluation.

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