Near-Perfect Long Reads Reveal Hidden Mosaicism Left Behind by CRISPR Gene Editing

CRISPR-Cas9 has transformed the life sciences by giving researchers a seemingly simple tool for cutting DNA at chosen locations, but a new study published in Genome Medicine delivers a sobering reminder that the aftermath of those cuts is far messier than standard screening methods suggest. A team led by Ida Höijer and Adam Ameur at Uppsala University, together with colleagues at Leiden University Medical Center, used an ultra-accurate targeted long-read sequencing approach to examine genome-edited zebrafish in unprecedented detail. What they found was widespread genetic mosaicism, with individual animals carrying a startling diversity of editing outcomes, including large structural rearrangements and off-target mutations that could silently pass to the next generation. The work, published open access on 15 September 2026, offers both a technical advance in how editing outcomes are measured and a cautionary message for anyone using CRISPR to create or validate animal models.

The core problem the researchers set out to solve is one that has dogged the genome-editing field for years. When Cas9 is introduced into a fertilized egg at the single-cell stage, as is routine in zebrafish studies, the enzyme may continue to cut DNA after the embryo begins to divide. Each subsequent cut-and-repair event creates a genetically distinct cell lineage, so the animal that develops is not a uniform carrier of one intended edit but a patchwork of different alleles distributed across its tissues. This phenomenon, known as mosaicism, is well recognized, yet characterizing it thoroughly has been difficult, particularly when the events involved are structural variants occurring at low frequency. Short-read sequencing struggles to resolve large insertions, deletions, and rearrangements, and amplification-based methods can distort the true proportions of variant molecules in a sample.

To overcome these limitations, the team turned to amplification-free targeted sequencing on the PacBio platform using the PureTarget approach, which enriches predefined genomic regions without polymerase chain reaction. Thirty samples from pooled zebrafish larvae and individual adult fish were sequenced successfully, yielding an average coverage of more than 1,100-fold across the target loci. Critically, the reads achieved a consensus accuracy of QV39, meaning fewer than one error per billion bases, which the authors describe as near-perfect. Every read originated from a unique DNA molecule, so the depth of coverage translated directly into an unbiased census of the alleles present. The two haplotypes of each target locus showed balanced depth of coverage, a sign that the method captures both maternal and paternal chromosomes without preference.

The contrast with conventional approaches was striking. When the researchers applied long-range PCR to the same samples before sequencing, the resulting data were skewed, with certain alleles overrepresented and others underrepresented relative to the amplification-free measurements. This matters because many laboratories still rely on PCR-based amplicon sequencing to verify their editing experiments, and the new results indicate that such workflows can systematically misrepresent the complexity of what is actually present in an edited animal. By removing the amplification step entirely and reading native DNA molecules directly, PureTarget preserves the true variant allele frequencies, allowing even rare structural variants to be detected and quantified with confidence.

The biological findings were equally significant. Analysis of individual founder fish, the F0 animals injected at the single-cell embryo stage, revealed extensive mosaicism. A single adult founder carried up to 18 distinct on-target editing events at the intended locus, and up to 11 different off-target events elsewhere in the genome. In other words, one fish that might superficially appear to carry a simple knockout mutation was in fact a living mosaic of dozens of different indels, deletions, inversions, and other rearrangements, each present in a different fraction of its cells. Many of these events would be invisible to standard genotyping PCR or short-read sequencing, which typically only confirms the presence or absence of the intended edit.

Perhaps the most consequential discovery concerned inheritance. When the founders were bred, several editing outcomes, including large structural variants and off-target mutations, were transmitted to the F1 generation. Among juvenile siblings derived from a single founder pair, the researchers identified as many as seven unique editing events. This confirms that mosaicism was present not only in the somatic tissues of the founders but also in their germ cells, meaning that different gametes carried different edited alleles. The practical implication is profound: some consequences of CRISPR-Cas9 editing may only become apparent in the second generation, so a founder that screens negative for off-target mutations in a blood or fin biopsy could still be silently transmitting unexpected variants to its offspring.

For the zebrafish community, which generates thousands of edited lines each year for studies of development, disease modeling, and gene function, these findings argue for more rigorous genotyping strategies. A line established from a mosaic founder may harbor multiple distinct alleles that segregate in subsequent generations, confounding phenotypic interpretation if researchers assume all carriers share an identical mutation. Structural variants that remove or invert large genomic segments can affect neighboring genes, and off-target mutations that hitchhike through the germline could introduce unrelated phenotypes. The study suggests that careful outcrossing and thorough molecular characterization of early generations remain essential, and that the depth and accuracy of the method used for that characterization determine what can actually be seen.

Beyond counting mutations, the platform also allowed the team to interrogate epigenetic signals embedded in the same sequencing data. PacBio reads carry kinetic information that can be used to infer DNA methylation, and the researchers analyzed 5-methylcytosine levels at CpG dinucleotides across the targeted regions in their edited samples. Notably, they did not observe altered 5mC CpG levels in the genome-edited fish compared with controls, providing some reassurance that Cas9 cutting at these loci did not perturb local methylation patterns. This ability to profile both genetic mosaicism and DNA methylation from a single amplification-free dataset illustrates the versatility of the approach for pre-defined genomic regions of interest.

The technical achievement rests on a combination of factors: amplification-free library preparation that avoids PCR-induced bias, targeted enrichment that concentrates sequencing capacity on the loci that matter, and the high intrinsic accuracy of PacBio HiFi reads, here pushed to exceptional consensus quality over the target regions. The authors note that the work was supported by a technology development project grant from SciLifeLab’s National Genomics Infrastructure, with library preparation performed in Uppsala and sequencing carried out at Clinical Genomics Uppsala. Some reagents were provided by PacBio, and one author received conference travel support from the company, disclosures that are stated in the paper. All zebrafish experiments were approved by the Uppsala University Ethical Committee for Animal Research.

The broader message for the genome-editing field is that the true complexity of CRISPR outcomes has likely been underestimated, not because the events are rare but because the tools used to detect them were not up to the task. As CRISPR-based therapies advance through clinical trials and edited animal models underpin countless studies, the ability to comprehensively profile mosaicism, structural variation, and off-target effects with single-molecule accuracy becomes a matter of scientific validity and safety. This study demonstrates that near-perfect long-read sequencing of targeted regions can provide that visibility, and it warns that what happens in the cutting bowl does not stay in the founder: some of CRISPR’s unintended edits can echo quietly into the next generation before anyone notices.

Subject of Research: Characterization of CRISPR-Cas9 editing outcomes and genetic mosaicism using near-perfect targeted long-read sequencing in zebrafish

Article Title: Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads

Article References: Höijer, I., van Schendel, R., Emmanouilidou, A., Östlund, R., Bunikis, I., Tijsterman, M., den Hoed, M., & Ameur, A. (2026). Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads. Genome Medicine, 18(1), Article 135. https://doi.org/10.1186/s13073-026-01772-1

Image Credits: AI Generated

DOI: 10.1186/s13073-026-01772-1

Keywords: CRISPR-Cas9, genome editing, genetic mosaicism, off-target mutations, structural variation, long-read sequencing, PacBio, PureTarget, zebrafish, DNA methylation, targeted sequencing, Genome Medicine