A comprehensive characterization of gene editing activity to support regulatory submissions: in silico off-target prediction, empirical off-target detection (GUIDE-seq/iGUIDE), confirmatory amplicon sequencing of nominated sites, and chromosomal translocation analysis via both ddPCR and amplicon sequencing, covering both on-target editing efficiency and unintended off-target/genome-integrity risk.

CRISPR-Cas9, CRISPR-Cas12a, CRISPR-CasX, base editing, prime editing, ARCUS, zinc finger nucleases (ZFNs), and TALENs.

In silico algorithms predict potential off-target loci based on sgRNA sequence homology to the genome, which we use alongside rhAmpSeq screening to prioritize candidate sites before moving to empirical confirmation.

GUIDE-seq and iGUIDE for genome-wide off-target site nomination in living cells, followed by targeted amplicon sequencing to confirm and quantify editing at the nominated sites. We also support Digenome-seq, SITE-seq, CIRCLE-seq, and custom assay development for programs that need a biochemical or alternative approach.

Yes. We run ddPCR-based translocation studies on the Bio-Rad QX200 platform for sensitive detection of translocation events between on- and off-target double-strand break sites, plus amplicon sequencing translocation analysis for high-specificity assessment across multiple site pairs.

Yes, 21 CFR Part 11 compliant, and run under CGMP/GLP-compliant conditions as appropriate to your study’s stage.

Because they answer different questions and neither alone is considered sufficient. In silico tools are fast and inexpensive but only surface predicted sites based on sequence homology, they capture no chromatin, repair-pathway, or actual nuclease-activity information. Empirical methods (biochemical or cell-based) experimentally detect cleavage events but each has its own blind spots. FDA’s current thinking is that sponsors should use multiple orthogonal methods rather than relying on any single approach, a position reinforced by FDA’s new draft guidance on NGS-based off-target safety assessment, which frames in silico and NGS-based methods as complementary tools rather than substitutes for one another.

Source: U.S. Food and Drug Administration (FDA)

In silico tools scan the reference genome computationally for sequence homology to your guide RNA or target sequence, useful for guide design and initial risk triage, but predictions only. Biochemical assays (e.g., CIRCLE-seq, SITE-seq, Digenome-seq) expose purified genomic DNA to your nuclease in vitro. While these methods are highly sensitive and comprehensive, they don’t capture chromatin structure or cellular repair pathways, so they can overestimate real-world editing activity. Cellular assays (GUIDE-seq, iGUIDE) edit living cells directly, capturing native chromatin and repair context, the most biologically relevant read on what will actually happen in your target cell type, at the cost of needing efficient delivery into that cell type. Which combination makes sense depends on your editor’s mechanism of action and development stage; FDA’s new NGS guidance recommends sponsors justify their choice of method(s) against the specific mechanism of their editor rather than defaulting to a one-size-fits-all approach.

Source: U.S. Food and Drug Administration (FDA)

FDA’s new guidance is specific about this: for every reported off-target site, the report should include genomic coordinate information, the number of mismatches/bulges relative to your guide RNA, PAM information (for CRISPR-Cas editors), whether the site is intergenic, exonic, or intronic, and a discussion of likely functional impact (e.g., effect on a nearby gene’s expression or splicing for exonic/intronic hits, or on regulatory regions for intergenic hits), along with a risk-assessment summary drawing on relevant published literature and gene-disease association data. We build reports to this structure, designed to be submission-ready rather than requiring reformatting before going into your IND.

Source: U.S. Food and Drug Administration (FDA)

FDA expects the full nonclinical off-target editing and chromosomal translocation package completed and submitted with the original IND application; however, studies accounting for human genetic variation may not be necessary with an original IND in some cases, such as an ultra-rare disease indication or treatment intended for a single patient. But sponsors don’t have to work this out alone first: FDA explicitly encourages discussing your off-target analysis strategy at an INTERACT or pre-IND meeting before you’ve generated the data, and even outlines the minimum information it wants to see in each (editor mechanism of action, planned nomination/confirmation/translocation methods at INTERACT; the fuller analysis-parameter and sequencing-strategy detail at pre-IND). Starting this conversation early enough to inform that meeting, rather than after, is the difference between a study plan FDA has already seen and one you’re defending for the first time.

Source: U.S. Food and Drug Administration (FDA)

Not automatically, but it’s a real trigger to check. FDA’s new guidance flags that additional off-target editing analysis may be needed if a manufacturing process change affects either the editor’s activity or the on-target editing rate, since either could shift the off-target site profile, and separately if new safety issues are identified during development. If a manufacturing change is planned, it’s worth looping in early so we can help assess whether it clears that bar.

Source: U.S. Food and Drug Administration (FDA)