// May 12, 2026 | American Society of Gene and Cell Therapy (ASGCT)
Comprehensive off-target assessment strategies for CRISPR-based genome editing therapeutics: integrating biochemical, bioinformatic, and unbiased approaches
Allie Crawley
Associate Director, Computational Biology, ElevateBio
Overview
Comprehensive assessment of gene editing specificity is key for developing genetic medicines. This presentation shows that a multi-layered nomination strategy improves detection of off-target sites across multiple editing modalities (nuclease, adenosine, and cytosine base editors) compared with any single method. Off-target profiles vary significantly by cell type, editing modality, and editor engineering, requiring evaluation in therapeutically relevant models using both male and female primary cells. This multi-assay approach is aligned with current regulatory guidance for the comprehensive assessment of genome editing safety. Our approach merges orthogonal nomination methods with deep sequencing validation and clinical variant analysis to systematically assess off-target editing risks for CRISPR therapeutics.
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Scientific Poster
Video Presentation & Transcript
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I’m Allie Crawley, and I’ll be sharing how ElevateBio integrates biochemical, computational, and cell-based approaches to assess off-target editing risks for CRISPR-based therapeutics. This helps partners build more comprehensive safety profiles for their target or candidate of interest.
So here at ElevateBio, we have produced base editing technologies across a large array of editing outcomes and have had to customize how we do off-target safety assessments for each of those tools. So looking at our comprehensive off-target workflow, we start with nominating off-target sites that have the potential for off-target editing.
We use knowledge-based annotations to understand some of the biological risk potential for those sites, and then we perform a confirmation assessment of editing to verify the rate of editing at all of the sites that were nominated.
Once we have positive sites, we’re able to do dose-response curves, we’re able to look in primary cells, and this gives us a more physiological look at the true off-target editing risk. We are able to do nomination of off-target approaches using both a biochemical and a homology-based approach.
We have customized those workflows to work with multiple different editing modalities, including nucleases, nickases, base editors, and RT editors. And we’ve paired our traditional biochemical and in silico-based approach with whole-exome sequencing that allows us to get to a 1% sensitivity allele frequency detection of off-target edits in an unbiased manner.
Once we’ve completed our full nomination for sites that have potential for off-target editing, we perform deep sequencing to really accurately estimate the empirically observed biological editing rates at these genomic sites. We are able to employ protein engineering to help reduce some of these off-target sites that get observed.
Through our comprehensive assessment, we’re able to accurately and comprehensively perform end-to-end safety evaluations across several different editing modalities. The framework I presented here gives partners a more complete view of their off-target editing risk, from nomination through confirmation and biological interpretation, so they can make stronger decisions as their CRISPR-based therapeutic candidates advance towards the clinic.
