Countagen Technology
Countagen Technology
Countagen’s patent-pending technology is being used to develop GeneAbacus™ solutions – gene editing analysis tools designed to accelerate genomic research, enabling researchers to move swiftly and confidently from gene edit to clone selection for functional analysis or to decide upon their editing strategy.
Gene editing has opened many opportunities to transform research, drug development and personalized medicine, enabling scientists to choose where to edit a gene in order to disable or activate a gene or to insert a completely new gene at a specific location. However, gene editing techniques are not perfect. To increase the chance of a successful project and avoid wasting time and resources, it is essential to determine the efficiency and specificity of an editing experiment before moving forward.
The people supporting product development at Countagen have decades of experience and knowledge in quantification and identification of nucleic acids, development of rapid, highly precise assays and delivery of user-friendly consumable solutions.
GeneAbacus assay principle
Free from the risk of PCR amplification bias, the GeneAbacus assay identifies desired edits and precisely quantifies editing efficiency.
Any CRISPR-based gene editing experiment can be analyzed within the working day, using standard laboratory instrumentation
- Analyze multiple samples per day
- Eliminate risk of amplification bias or cross-contamination – PCR independent
- Avoid risk of bottlenecks – no need to outsource or optimize analytical conditions
- Integrate easily into a standard laboratory workflow
- No method optimization or amplicon purification
- No specialized training – uses standard laboratory equipment such as fluorescence microscopy and PCR cyclers
- Ensure consistency – automated image analysis of digitized fluorescent spots
- Obtain quantitative data – number of spots relate to gene editing efficiency
GeneAbacus assay principle
Free from the risk of PCR amplification bias, the GeneAbacus assay identifies desired edits and precisely quantifies editing efficiency.
Any CRISPR-based gene editing experiment can be analyzed within the working day, using standard laboratory instrumentation
- Analyze multiple samples per day
- Eliminate risk of amplification bias or cross-contamination – PCR independent
- Avoid risk of bottlenecks – no need to outsource or optimize analytical conditions
- Integrate easily into a standard laboratory workflow
- No method optimization or amplicon purification
- No specialized training – uses standard laboratory equipment such as fluorescence microscopy and PCR cyclers
- Ensure consistency – automated image analysis of digitized fluorescent spots
- Obtain quantitative data – number of spots relate to gene editing efficiency
Countagen technology step-by-step
Countagen technology can be divided into 7 distinct steps, all performed within as little as 5 hours. The assay begins with extracted DNA (Step 0) and concludes with delivery of actionable results i.e. editing efficiency and specificity (Step 6).
step 0.
Design and develop the timeplan for a gene editing experiment
Order a GeneAbacus kit. Countagen specialists will create custom-made kits to ensure delivery, ready-to-use and on time, for gene editing analysis.
Perform the gene editing experiment.
Extract gDNA using a method of choice. The assay is compatible with DNA extracted from wild type and edited populations of cells, tissues or organisms such as Drosophila and Zebrafish.
step 1.
DNA Fragmentation
The first reaction step of the assay is the fragmentation of extracted DNA.
Step 2.
Probing Target DNA
Tailored linear padlock probes against a reference and the modified genes are used to target the sequences of interest.
Only in the event of a perfect match, will a DNA ligase seal the nick and lock the padlock probe onto the DNA target strand. The specificity of the padlock probes and the DNA ligase enables discrimination of single nucleotide polymorphisms (SNPs) and INDELs.
Step 3.
Signal Generation
Circularized strands containing the target region are amplified via Rolling Circle Amplification (RCA).
In RCA, the circular template is replicated as a long single-stranded DNA concatemer that spools off when amplified by a strand-displacing DNA polymerase. The resulting concatemeric amplicon consists of hundreds to thousands of repeats of the circular template.
Step 4.
Labeling
RCA amplicons, also termed RCA products, are labeled with sequence-specific fluorescent probes.
The single-stranded RCA product collapses into a DNA micro-ball with minimal inter-molecular interactions due to the high negative charge stemming from the phosphate backbone. This single-molecule integrity makes RCA ideal for subsequent digital nucleic acid quantification.
Step 5.
Imaging
RCA products are easily detected using standard fluorescence microscopy at low magnification (20X).
Countagen technology enriches all RCA products from solution into one field of view, making imaging, quantitation and analysis extremely simple. As shown in the scheme, RCA products are detected as bright fluorescent dots, where each dot represents a single DNA target from the beginning. This is the strength of RCA, being inherently digital without the need for compartmentalization as in digital PCR.
step 6.
Image Analysis
The final step is image analysis through GeneAbacus Image Analyzer (GAIA) software.
GAIA is provided as a downloadable file, available upon receipt of an email confirming a kit purchase. An email address for the researcher performing the analysis must be provided when ordering.
GAIA enables simple analysis of acquired images via an intuitive graphical user interface. The software automatically identifies and counts fluorescent spots which relate directly to the editing efficiency of the experiment. Besides a drop-off rate, the assay also enables precise quantification of desired mutations through knock-ins, base editing and prime editing by introducing additional probes.
step 0.
Design and develop the timeplan for a gene editing experiment
Order a GeneAbacus kit. Countagen specialists will create custom-made kits to ensure delivery, ready-to-use and on time, for gene editing analysis.
Perform the gene editing experiment.
Extract gDNA using a method of choice. The assay is compatible with DNA extracted from wild type and edited populations of cells, tissues or organisms such as Drosophila and Zebrafish.
step 1.
DNA Fragmentation
The first reaction step of the assay is the fragmentation of extracted DNA.
Step 2.
Probing Target DNA
Tailored linear padlock probes against a reference and the modified genes are used to target the sequences of interest.
Only in the event of a perfect match, will a DNA ligase seal the nick and lock the padlock probe onto the DNA target strand. The specificity of the padlock probes and the DNA ligase enables discrimination of single nucleotide polymorphisms (SNPs) and INDELs.
Step 3.
Signal Generation
Circularized strands containing the target region are amplified via Rolling Circle Amplification (RCA).
In RCA, the circular template is replicated as a long single-stranded DNA concatemer that spools off when amplified by a strand-displacing DNA polymerase. The resulting concatemeric amplicon consists of hundreds to thousands of repeats of the circular template.
Step 4.
Labeling
RCA amplicons, also termed RCA products, are labeled with sequence-specific fluorescent probes.
The single-stranded RCA product collapses into a DNA micro-ball with minimal inter-molecular interactions due to the high negative charge stemming from the phosphate backbone. This single-molecule integrity makes RCA ideal for subsequent digital nucleic acid quantification.
Step 5.
Imaging
RCA products are easily detected using standard fluorescence microscopy at low magnification (20X).
Countagen technology enriches all RCA products from solution into one field of view, making imaging, quantitation and analysis extremely simple. As shown in the scheme, RCA products are detected as bright fluorescent dots, where each dot represents a single DNA target from the beginning. This is the strength of RCA, being inherently digital without the need for compartmentalization as in digital PCR.
step 6.
Image Analysis
The final step is image analysis through GeneAbacus Image Analyzer (GAIA) software.
GAIA is provided as a downloadable file, available upon receipt of an email confirming a kit purchase. An email address for the researcher performing the analysis must be provided when ordering.
GAIA enables simple analysis of acquired images via an intuitive graphical user interface. The software automatically identifies and counts fluorescent spots which relate directly to the editing efficiency of the experiment. Besides a drop-off rate, the assay also enables precise quantification of desired mutations through knock-ins, base editing and prime editing by introducing additional probes.