Surreal illustration of DNA strands and CRISPR-Cas9 editing tools.

CRISPR Made Easier: How New Testing Methods Can Refine Gene Editing

Kai Mendoza in Science & Nature August 2025 • 4 min read.

"Discover how innovative qPCR and dPCR techniques are revolutionizing CRISPR-Cas9 gene editing, providing faster, more accurate results for groundbreaking research."

CRISPR-Cas9 technology has revolutionized gene editing, offering unprecedented flexibility in creating targeted mutations. Yet, the complexity of genetic outcomes, especially in animal models, often presents significant analytical challenges. Traditional methods struggle to keep pace with the diverse range of edits that can occur, leading to inefficiencies and potential inaccuracies in research.

In a recent study, scientists have introduced innovative approaches using quantitative PCR (qPCR) and digital PCR (dPCR) to enhance the detection and quantification of CRISPR-Cas9 mediated gene edits. These methods promise to streamline the analysis process, providing rapid and precise measurements of on-target editing events in vivo. By improving the accuracy and speed of CRISPR analysis, researchers can accelerate their work, reduce costs, and gain deeper insights into genetic modifications.

These advancements address critical limitations in current genomic research, offering a more reliable way to assess the efficacy and specificity of CRISPR edits. This article explores how these new methods are transforming gene editing, making it more accessible and efficient for researchers worldwide.

Enhanced CRISPR Detection with qPCR and dPCR

Surreal illustration of DNA strands and CRISPR-Cas9 editing tools.

The study focuses on refining methods to detect and quantify the various genetic outcomes of CRISPR-Cas9 editing, particularly in mouse models. Traditional techniques, such as Sanger sequencing and probe competition assays, often fall short in capturing the full spectrum of allelic diversity. These methods can be time-consuming, costly, and may miss low-frequency variants, complicating the analysis of CRISPR-edited organisms.

To overcome these limitations, the researchers developed and validated specific qPCR and dPCR methods. These techniques offer several advantages, including higher sensitivity, increased accuracy, and the ability to detect a wide range of editing events in a single assay. By using locked nucleic acid (LNA) probes combined with internal reference probes, the new assays accurately measure editing rates and detect on-target CRISPR-Cas9 gene editing in blastocysts with sensitivity comparable to PCR-clone sequencing.

Key benefits of the new qPCR and dPCR methods:

Increased Sensitivity: Detects low-frequency editing events that traditional methods may miss.
Improved Accuracy: Provides more precise quantification of editing rates.
Higher Throughput: Allows for rapid analysis of multiple samples.
Cost-Effective: Reduces the need for extensive sequencing.

The qPCR and dPCR assays were rigorously tested and validated using samples from CRISPR-edited mice. The results demonstrated that the LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo, testing RNA guides, and detecting recombinant mutations. These methods offer a significant improvement over existing techniques, providing researchers with a more reliable and efficient way to assess CRISPR-Cas9 editing outcomes.

The Future of CRISPR Analysis

The development of these refined qPCR and dPCR methods marks a significant step forward in CRISPR-Cas9 technology. By providing faster, more accurate, and more sensitive detection of gene editing events, these techniques empower researchers to refine their approaches and accelerate the pace of discovery. As gene editing continues to advance, such innovations will be essential for unlocking its full potential and ensuring reliable, reproducible results. The internally referenced LNA probe system described in the research can be used as a founder-generation screening tool, enabling rapid genotyping of recombinant and indel alleles, thereby streamlining research processes.

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

This article is based on research published under:

DOI-LINK: 10.1534/g3.117.300123, Alternate LINK

Title: Single-Step Qpcr And Dpcr Detection Of Diverse Crispr-Cas9 Gene Editing Events In Vivo

Subject: Genetics (clinical)

Journal: G3 Genes|Genomes|Genetics

Publisher: Oxford University Press (OUP)

Authors: Micol Falabella, Linqing Sun, Justin Barr, Andressa Z Pena, Erin E Kershaw, Sebastien Gingras, Elena A Goncharova, Brett A Kaufman

Published: 2017-10-01

Everything You Need To Know

How do qPCR and dPCR improve CRISPR-Cas9 gene editing?

The innovative qPCR and dPCR techniques enhance CRISPR-Cas9 gene editing by offering precise and rapid detection of diverse genetic modifications. These methods streamline the analysis process, providing rapid and precise measurements of on-target editing events in vivo. This results in improved accuracy and speed of CRISPR analysis, allowing researchers to accelerate their work, reduce costs, and gain deeper insights into genetic modifications.

What are the limitations of traditional CRISPR analysis methods compared to qPCR and dPCR?

Traditional methods like Sanger sequencing and probe competition assays often struggle to capture the full spectrum of allelic diversity, especially in animal models. They can be time-consuming, costly, and may miss low-frequency variants. In contrast, qPCR and dPCR offer higher sensitivity, increased accuracy, and the ability to detect a wide range of editing events in a single assay, addressing the limitations of traditional techniques.

What are LNA probes and how do they enhance qPCR and dPCR assays in CRISPR analysis?

LNA probes are locked nucleic acid probes used in conjunction with qPCR and dPCR assays to enhance the detection and quantification of CRISPR-Cas9 mediated gene edits. These probes, combined with internal reference probes, accurately measure editing rates and detect on-target CRISPR-Cas9 gene editing in blastocysts with sensitivity comparable to PCR-clone sequencing. The internally referenced LNA probe system can be used as a founder-generation screening tool, enabling rapid genotyping of recombinant and indel alleles, thereby streamlining research processes.

What are the key advantages of using qPCR and dPCR methods for CRISPR analysis?

The key advantages of using qPCR and dPCR methods include increased sensitivity to detect low-frequency editing events, improved accuracy in quantifying editing rates, higher throughput for rapid analysis of multiple samples, and cost-effectiveness by reducing the need for extensive sequencing. These benefits collectively provide researchers with a more reliable and efficient way to assess CRISPR-Cas9 editing outcomes, particularly in complex genetic scenarios such as those found in mouse models. This results in time savings, reduced costs, and more comprehensive insights into genetic modifications.

What are the implications of qPCR and dPCR methods for the future of CRISPR analysis and gene editing research?

The qPCR and dPCR methods have a broad implication for future CRISPR analysis. By providing faster, more accurate, and more sensitive detection of gene editing events, these techniques empower researchers to refine their approaches and accelerate the pace of discovery. As gene editing continues to advance, such innovations will be essential for unlocking its full potential and ensuring reliable, reproducible results. These methods can be used as a founder-generation screening tool, enabling rapid genotyping of recombinant and indel alleles, thereby streamlining research processes and potentially leading to the development of new gene therapies and biotechnologies.