Our SureGuide OnTarget Platform is a robust tool for evaluating off-target and on-target effects based on high-throughput sequencing in CRISPR-Cas applications. It possesses a remarkable capability: even when the frequency of a specific off-target mutation is as low as 0.1%, our methodology can accurately detect and analyze it.

Overview of Our Platform

The CRISPR-Cas system, while groundbreaking, faces a notable limitation: the generation of redundant DNA mutations at sites beyond the intended target. Recent studies have unequivocally revealed that CRISPR/Cas9 exhibits pronounced off-target effects, wherein non-specific cleavage occurs, inducing mutations at sites in the genome unrelated to the targeted region. This phenomenon introduces uncertainty into research outcomes, necessitating a substantial increase in research efforts. Consequently, this issue has significantly hampered the widespread application of Cas9.

To address this challenge, our services offer global off-target site detection using advanced next-generation sequencing platforms. Leveraging SureGuide OnTarget Platform, a genome-wide method employing high-throughput sequencing technology, we provide a sensitive approach. This platform maintains heightened sensitivity for detecting low-frequency mutations even in instances of low transfection efficiency, thereby enhancing overall detection accuracy.

The underlying principle involves the utilization of a short double-stranded oligonucleotide tag to uniquely label breaks induced by CRISPR-Cas, encompassing both on-target and off-target sites. After this tagging process, we employ high-throughput sequencing focused on the gene region housing the tag. The final step involves a sophisticated bioinformatics analysis, pinpointing the location of off-target mutations and determining their frequency.

Workflow of SureGuide OnTarget Platform

CD Genomics has been committed to making a difference in your off-target detection services through high-throughput sequencing platforms to support CRISPR optimization services during gene editing. The importance of off-target detection directly determines the success of gene editing. Our Sequencing-based CRISPR off-target detection service process is shown in the figure below, and a professional team of experts under strict QC standards operates the whole process.

01. Sequencing sample requirements

• Cell lines after gene editing (frozen or in culture dishes)

02. Library construction and NGS

• NGS-based Illumina / MGI platform

03. Bioinformatics Analysis

• Mutation detection
• Detection of sgRNA homology region
• Detection of potential off-target sites in the PAM region
• Statistics on-target indel, off-target rate and editing rate

Advantages of SureGuide OnTarget Platform

Our SureGuide OnTarget Platform boasts a superior signal-to-noise ratio, necessitating fewer sequencing reads, ensuring heightened sensitivity in identifying genome-wide off-target sites. These characteristics collectively position it as a superior performer compared to existing cellular or biochemical methods for genome-wide CRISPR/Cas9 off-target mutation identification.

• High Utility: Reflects both on-target and off-target conditions in CRISPR gene editing within eukaryotic cells, facilitating safety and efficacy evaluations.
• High Throughput: Enables simultaneous detection of multiple samples for on- and off-target assessment, optimizing label design to reduce actual sequencing reads.
• High Accuracy: Results are largely verifiable, enhancing the reliability of the findings.
• High Sensitivity: Achieves efficient detection of low-frequency off-target sites, capable of identifying mutations as rare as 0.1%.
• Combined Use with Amplicon Sequencing: Enhances assay reliability when used in conjunction with SureGuide OnTarget Platform.
• Highly Cell-Adaptable: Adaptable to diverse cell types, ensuring versatility in experimental setups.
• Short Cycle Time: Efficient processes result in a shorter turnaround time, providing raw data and a comprehensive visualization in the completion report.

Sample Requirements

Host cell + Cas9 +sgRNA (Plasmid, RNP or RNA format) or Cryopreserved cells: Cells are transfected with Cas9, sgRNA and dsODN tags. At least 4x10⁶ cells