Streamlining routine CRISPR validation with Oxford Nanopore sequencing

As CRISPR-based editing becomes increasingly integral to biopharma workflows, particularly for gene knockout generation, functional screening, targeted gene correction, and next-generation cell and gene therapy development, the need for scalable, cost-effective validation methods is growing. Guide RNA (gRNA) validation for knockouts typically relies on assessing insertions and deletions (indels) at the target site; an essential quality control step to confirm gene disruption.

Meeting the need for scalable indel screening

Traditionally, researchers have relied on Sanger sequencing-based methods such as Tracking of Indels by Decomposition (TIDE)¹ and Inference of CRISPR Edits (ICE)² for this task. However, these approaches are limited by throughput, turnaround time, and inflexibility in handling long amplicons, making them less suitable for routine or high-volume use.

McFarlane, Polanco, and Bogema evaluated an Oxford Nanopore-based alternative as a solution for routine gRNA validation, pairing long amplicon sequencing with the analysis software CRISPResso2³: a bioinformatics tool for assessing PCR amplicons spanning gRNA target sites for indel analysis. The results offer a compelling case for replacing Sanger in this critical application.

Delivering concordant results but with added flexibility

To test the performance of Oxford Nanopore sequencing for indel detection, the research team targeted the myostatin (MSTN) gene in sheep and horse fibroblasts with two CRISPR-Cas9 gRNAs⁴. After extracting DNA from the cells and amplifying the target region with PCR, they either sequenced the resulting >600 bp amplicons with Oxford Nanopore technology or sent them for Sanger sequencing at the Australian Genome Research Facility.

For Oxford Nanopore sequencing, the team prepared the PCR products using the Native Barcoding Kit and loaded the libraries on MinION Flow Cells to run them on a GridION device. The nanopore data was analysed with a nanopore-compatible version of CRISPResso2 (nCRISPResso2), and the Sanger data was analysed using TIDE and ICE.

The researchers found that indel frequencies obtained from Oxford Nanopore sequencing closely mirrored those from both Sanger-based approaches, with particularly strong alignment between nCRISPResso2 and ICE. The authors stated: ‘Notably, nCRISPResso2 exhibited closer alignment with ICE results than with TIDE, or even between TIDE and ICE themselves’. The top five most common indel outcomes also appeared in the same order across all three methods, further reinforcing the reliability of the Oxford Nanopore approach.

By pairing in-house long amplicon sequencing with a bioinformatic tool, the researchers showed that Oxford Nanopore sequencing could not only match the accuracy of established methods but also unlock additional benefits such as higher throughput (Figure 1).

Figure 1. Oxford Nanopore sequencing delivers scalable and accurate in-house CRISPR indel analysis that goes beyond the limits of traditional methods.

Suitable for high-throughput biopharma workflows

Although the published results focus on two gRNAs, the researchers also applied their method to an additional 14 gRNAs within the same nanopore sequencing run, illustrating the scalability and efficiency of the approach. This kind of multiplexing is particularly valuable in biopharma settings where multiple targets are routinely screened in parallel. As the authors highlight, ‘multiplexing significantly reduces costs, enhancing scalability and affordability.’

Beyond routine CRISPR validation, the benefits of long amplicon sequencing apply to a wide range of use cases where Sanger is traditionally used. These include antibody screening, vector construct verification, and cell line development, all of which can benefit from the real-time, flexible, and scalable nature of Oxford Nanopore workflows.

Encouraging wide adoption in routine gene editing

This study demonstrates how Oxford Nanopore sequencing can serve as a reliable, flexible alternative to traditional methods for gRNA validation and indel analysis, delivering results that are highly concordant with established tools, while enabling greater throughput and in-house control. Teams can move quickly from sample to insight, without the need for core sequencing facilities.

As the authors conclude: ‘We hope this study encourages the adoption of nCRISPResso2 by fellow genome editors to streamline indel analyses and reduce costs.’

With clear advantages in turnaround time, compatibility with long targets, and potential for broad application across molecular biology and biopharma, Oxford Nanopore sequencing is positioned to become a core tool for routine amplicon analysis.