In genome editing, precision is the goal, but biology has a way of introducing variation of its own. Genetic mosaicism occurs when different genetic variants coexist within the same organism, often as an unintended byproduct of CRISPR-Cas9 editing. Because CRISPR can remain active for several cell divisions, a single edited embryo can develop into a patchwork of genotypes, each carrying unique insertions, deletions, or rearrangements.
This variability matters. Mosaicism complicates how scientists interpret editing outcomes and raises questions about which alleles might pass to offspring. And when each cell or animal model can cost tens of thousands of dollars to create, researchers need to know they’ve faithfully edited the genes they intended, without introducing off-target changes. That’s why it’s critical to have a high-sensitivity assay that can accurately characterize every edit within a model, from rare variants to large structural changes, without the distortions of amplification. That’s where PureTarget with HiFi sequencing comes in.
Capturing the complete picture of mosaicism
Mosaicism inherently presents a persistent technical challenge: the more diverse the edits, the harder they are to detect. Standard PCR and short-read sequencing methods often skew allele frequencies or miss large structural variants entirely, leaving much of this genetic complexity unresolved. To break through the limits of traditional approaches, researchers from Uppsala University turned to amplification-free PureTarget in their new pre-print, “Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads.” This study shows that PureTarget can uncover the complete landscape of CRISPR edits, from rare alleles to heritable variants, setting a new standard for how mosaicism is measured and understood in genome editing.
Setting a new standard with PureTarget
In the paper, the Uppsala team used a custom PureTarget panel to characterize CRISPR-Cas9 edits in 32 founder (F0) and offspring (F1) zebrafish samples, a widely used model for developmental and genome editing research. Four on-target loci (ldrla, nbeal2, sh2b3, ywhaqa) and three off-target sites (~5 kb each) were enriched with Cas9 and sequenced on a single Revio SMRT Cell with SPRQ chemistry.
Despite working with partially degraded DNA that had been stored for several years, these samples produced high-quality data, achieving an average 1,168× target coverage and QV39 read accuracy, roughly one error per 8,000 bases. These “near-perfect long reads” offered a single-molecule view of virtually every CRISPR outcome. By sequencing each molecule directly, PureTarget avoided amplification bias entirely. As a result, the team could confidently detect variants down to 1% frequency and accurately quantify mosaic alleles in both founders and offspring.
When compared to long-range PCR (LR-PCR), the performance of PureTarget is striking. Where LR-PCR data shows skewed allele frequencies, PureTarget maintains a consistent 50:50 distribution at heterozygous sites. This balance means no allele is overrepresented or lost, a crucial advantage for studying mosaicism and other low-frequency events.
Frequencies of reference (blue) and alternative (red) alleles at single nucleotide variant (SNV) positions in heterozygous target regions for F1 zebrafish. The bars to the left show allele frequencies for long-range PCR (LR-PCR) while the bars to the right show allele frequencies for PureTarget in the same samples. The minor allele frequencies obtained by PureTarget are close to 50% while LR-PCR results show skewed frequencies of the two haplotypes. The colors below the bars indicate which of the target sites were examined. The target sites have been detailed below each bar. Reproduced with permission from Höijer et al. (2025), Figure 2b.
What mosaicism across generations means for genome editing
Importantly, the researchers found that mosaicism was pervasive. Individual founder fish carried 7–18 distinct on-target variants and up to 11 off-target alleles within a single organism. Several of these edits, like a 1,053 bp deletion at sh2b3 and a 3 bp deletion at ywhaqa, were inherited by F1 fish, indicating that this mosaicism extended into germ cells. For anyone studying germline genome editing, that finding is striking. It means unintended edits can persist through generations, underscoring the need for precise, unbiased sequencing to verify what’s really happening at both on- and off-target sites. Because PureTarget preserves native methylation signals, the team also examined 5mC CpG methylation across edited and control zebrafish. They found no methylation differences in coding regions, but the ability to integrate epigenetic information into the same dataset opens new doors. It’s now possible to connect editing outcomes with their impact on methylation signatures, which can shape gene expression, all without extra assays or altered workflows.
How PureTarget makes it possible
Behind these results is a method purpose-built for precision. PureTarget combines Cas9-based enrichment, amplification-free library prep, and HiFi long-read sequencing to deliver exceptionally accurate, single-molecule data.
With custom panels, researchers can design panels spanning 20–500 kb and capture difficult regions like repeat expansions or GC-rich loci with consistent coverage. PureTarget achieves this while maintaining native methylation profiles and allowing high multiplexing and automation on the Revio system, or flexible batching on the affordable Vega benchtop system.
By eliminating PCR, PureTarget provides unbiased haplotype representation, even in heterozygous or mosaic samples, and enables the detection of every variant type, including large structural rearrangements. PureTarget enables a level of clarity that older approaches can’t match, now allowing researchers to consolidate several methods into a single assay.
Redefining the future of genome editing with HiFi sequencing
This study shows that PureTarget with HiFi sequencing can reveal the complete range of genome editing outcomes in a way that other methods can’t. This is significant as genome editing applications expand from basic research into applied contexts where understanding every outcome, not just the intended one, is essential.
In the words of the authors, PureTarget “enables efficient, accurate, and unbiased analysis of the full spectrum of genetic mosaicism in a sample.” This is a powerful proof point for how HiFi sequencing continues to shape the future of genome editing research, one accurate read at a time.
To explore how PureTarget can help you capture the complete picture of your genome editing outcomes, visit the PureTarget product page or download the PureTarget Custom Panels Technical Note.