Out-of-the-Box Gene Editing Analysis with Countagen

- It’s great to meet you all today. Before we get into the technical details, perhaps you could tell me about Countagen’s overall goal within the gene-editing space?

Our major focus is the demand for quality control to accurately measure the efficiency and specificity of gene-editing workflows. We want to simplify and accelerate this step for any researcher that uses gene-editing tools.

Accelerating the transition from gene editing to functional analysis

- What problem does Countagen intend to solve within gene editing?

CRISPR-Cas and related gene-editing tools continue to push the limits of what is possible within research and development, but quantifying editing efficiency and identifying cells with the intended edits remains a bottleneck in many gene-editing workflows.

We address this bottleneck by providing highly accurate tools to rapidly quantify gene-editing efficiency with single-nucleotide resolution. Our newly-launched product GeneAbacus can be used in-house in any molecular biology laboratory using standard equipment, and it provides quantitative data from a gene-editing experiment within just five hours.

- The need to measure gene-editing efficiency has existed since these tools emerged. How does Countagen’s approach differ from the methods that are already out there?

It’s true that the need to quantitatively assess gene-editing outcomes is not new. However, irrespective of which gene-editing modality is used, the standard approaches to quality control all require PCR amplification as the first step.

Any molecular biologist will know that primer optimisation can be tedious and very time-consuming, and PCR introduces the risk of amplification bias. On top of that, PCR-based methods lack single-nucleotide discrimination power, and thus require downstream multi-step sequencing workflows that often demand the user to access and use complex bioinformatic tools.

Locking down the target with single-nucleotide precision

- So, how does the GeneAbacus technology work?

In simple terms, the GeneAbacus workflow provides a count of edited and unedited cells in a pool or population, following a gene-editing experiment.

The assay begins with purified genomic DNA from the cells of interest. This is often a human or mouse cell line that has undergone CRISPR-Cas editing, but the assay should work even if other gene-editing modalities are used. We use padlock probes to detect parental (i.e., wild-type, unedited) or edited gene sequences in the genomic DNA.

The probes are linear, single-stranded oligonucleotides that are custom-designed to contain either the parental or the intended edited DNA sequence targets whereby probes to detect the intended edit contain the exact same sequence region as the guide RNA used to introduce the edit.

In the event of a perfect sequence match between the genomic DNA and a probe, a high-fidelity ligase will join the ends of the probe, effectively locking it on its target. Single-nucleotide precision is ensured by the fact that ligation will only occur when there is a perfect sequence match between the genomic DNA and the probe. A probe that specifically detects an endogenous gene is also included as an assay internal control for each GeneAbacus run. The parental, edit and assay reference probes have different barcodes that are amplified by isothermal rolling circle amplification (RCA), which is catalysed by a proprietary enzyme mix.

The RCA step generates individual long, barcoded single-stranded DNA molecules of approximately one micron in diameter. These molecules contain multiple repeats of the probe sequence and are subsequently labelled with different fluorescent dyes based on their barcodes and detected as single fluorescent spots with widefield fluorescence microscopy. This allows digital quantification of each single molecule amplification of the parental, intended edit and assay reference sequence.

- How are the fluorescently-labelled spots used to generate quantitative data about the number of edited and unedited cells?

In addition to the GeneAbacus reagents, we have developed and patented a dedicated readout technology that concentrates the fluorescent spots in a single field of view. This processing step allows for rapid imaging with 20x magnification.

Our companion image analysis software, GeneAbacus Image Analyzer (GAIA), then analyses these images by quantifying the number of spots in each fluorescence channel to yield calculations of the relative numbers of cells containing parental or edited DNA (% gene editing efficiency). As mentioned earlier, this makes GeneAbacus an all-in-one solution and the entire workflow can be completed within five hours, which leaves the researcher with a lot more walkaway time than the current methods.

Faster and simplified clonal selection with unmatched specificity

- Can you describe a typical use case for GeneAbacus?

A customer typically approaches us when they face bottlenecks in analysing their gene-editing experiment and are seeking a reliable and quick method to detect their desired edit in heterogeneous pools of cells or single cell clones.

In this case, the customer provides us with the parental and intended edited gene sequences, which we use to design the respective probes. In this way, each GeneAbacus assay is customised to the customer’s gene-editing experiment.

The data generated by GeneAbacus provides an accurate percentage of non-edited cells as well as the percentage of cells that harbour the intended edit. In the case of a heterozygous clone (i.e., the intended edit is present on one allele in a diploid organism), the editing efficiency would be 50%, while 100 % editing efficiency would indicate a homozygous clone.

It is important to note that many researchers face challenges in identifying homozygosity or heterozygosity in clones, especially if gene editing results in hemizygous cells, i.e., where one of the target alleles is completely knocked out. With Sanger sequencing, such clones may appear homozygous when they are in fact hemizygous. Because we can quantify the frequency of the intended edit, we solve that challenge. GeneAbacus users find it much faster and simpler than the current multi-step workflows to analyse pools and perform subsequent clonal selection.

- How precise is the GeneAbacus assay, and how does this compare to standard methods?

We have performed extensive experiments on the specificity of our padlock probes against a panel of human genes, whereby the probes were designed to be a perfect sequence match for the target gene or designed to contain a single nucleotide mismatch in the first position at the 3’ end.

These studies revealed that a single base mismatch prevents the ligation of the probe, and thus no template is formed for RCA. The resulting signal from these mismatching probes was less than 5 % of the signal counts from a perfectly-matched probe. These studies further confirm the single-nucleotide discrimination power of the assay, and within the field, this level of precision and specificity is unmatched.

As a side note, we have also demonstrated that GeneAbacus is compatible with gene regions with high GC content (66-84 %), without any loss of specificity and need for assay optimisation, such as annealing temperature and time, or redesign of the probes.

In addition, benchmarking studies against amplicon sequencing (NGS + analysis with CRISPResso2) in a set of 26 one-cell stage mouse embryos revealed excellent correlation of R² = 0.9498 between the two methods.

High-throughput version in the pipeline

- As I understand it, the current assay can detect two outcomes. Is it possible to optimise the GeneAbacus technology for multiplex detection without losing the current resolution?

The current version of GeneAbacus can process 32 samples in a run and is optimised for two customised probes as well as a control probe that specifically detects an assay reference gene, i.e., an unrelated gene that is not expected to be edited. This means that the assay can detect the parental sequence and the intended edited sequence, or two possible edited sequences.

We are in the process of developing a higher throughput version to meet the demand of analysing > 48 gene-edited samples in a run. This version will feature compatibility with rapid DNA extraction workflows and multi-well plate formats for easy integration into the current workflow. We are expecting to have this ready in the near future and are further leveraging the versatility of our technology to address more needs within the market, such as Indel sequence information, as we continue our mission to simplify gene-editing workflows.

- Much of the current hype around gene editing is focused on therapeutic applications. What fields is GeneAbacus currently used in, and how do you see this developing in the future?

GeneAbacus primarily serves academic laboratories that routinely use CRISPR-Cas editing to study gene functions in cells or model organisms. These researchers typically study the underlying biological mechanisms and/or carry out early-stage research to find novel therapeutic targets. Given the power of GeneAbacus to discriminate between homo- and heterozygosity, we see great potential with genotyping applications, and this is something we are exploring.

Regarding species, we can fully support human and mouse cell lines, including stem cell-derived cell lines, as well as mouse embryos. We’ve also successfully tested GeneAbacus on genomic DNA isolated from edited zebrafish embryos and fruit flies.

While we haven’t explored it yet, we see huge potential for GeneAbacus within agricultural research, particularly as a tool to simplify crop breeding.

Together, we innovate: adapt GeneAbacus to your genome editing research

- Since the assay input is purified genomic DNA, do you expect that GeneAbacus should work with any species?

Yes, theoretically, as long as the DNA quality is high and we/the customer can identify an assay reference gene that is very stable and not likely to be targeted by the gene-editing strategy.

Most current users of GeneAbacus are working with human or mouse cells, but we are also working on a case-by-case basis with researchers studying other species.

Although Countagen was officially founded in 2020, we have actually been working with the GeneAbacus technology for a decade at Science for Life Laboratory in Stockholm, and we have tried many things, so we welcome researchers from all disciplines to get in touch with us if they are interested in trying the assay for a species not mentioned here!

To learn more about Countagen and to find out how GeneAbacus can speed up your research, get in touch with the team for an informal chat here or send an email to info@countagen.com.

References

1. Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary BP, Landegren U. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science. 1994 Sep 30;265(5181):2085-8.
2. Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet. 1998 Jul;19(3):225-32.