At CD Genomics, our CRISPR Off-Target Validation Sequencing harnesses advanced multiplex PCR technology combined with probe hybridization panels and cutting-edge next-generation sequencing (NGS) to deliver unparalleled precision in identifying and validating off-target edits across a multitude of loci in a single assay—offering researchers heightened specificity and efficiency in their CRISPR endeavors.

Overview of CRISPR off-target validation sequencing

CRISPR off-target effects occur when the CRISPR-Cas system edits unintended regions of the genome. These off-target edits arise due to sequence similarities between the target DNA and other regions, causing the CRISPR mechanism to occasionally cut in the wrong place. Identifying and validating these off-target events is critical for ensuring the safety and accuracy of CRISPR-based applications.

To detect off-target sites, methods like Whole Genome Sequencing (WGS), GUIDE-Seq, and Circle-Seq are commonly used. These techniques help pinpoint potential off-targets across the genome. However, once these sites are identified, they need to be thoroughly validated to determine the actual editing frequency, which is a crucial but time-intensive part of the process. At CD Genomics, we offer advanced off-target validation sequencing, helping researchers confirm off-target events with high precision across multiple sites. This capability makes us one of the few companies equipped to handle such complex validation workflows efficiently.

The workflow of CRISPR off-target validation sequencing

The workflow for CRISPR off-target validation sequencing involves a systematic approach that ensures accurate detection and quantification of off-target edits. This process begins with careful sample preparation, followed by the construction of amplicon libraries using a proprietary probe hybridization panel. The libraries are then subjected to next-generation sequencing (NGS) to capture comprehensive data, which is subsequently analyzed using advanced bioinformatics tools to provide reliable insights into editing frequencies.

1. Sample Preparation

• Collect genomic DNA samples from the target organism.
• Ensure DNA quality and concentration meet the required standards (≥10 ng).

2. Library Construction

• Design a custom probe hybridization panel tailored to the target and off-target sites.
• Generate amplicon libraries through a two-step PCR process, utilizing our proprietary technology to minimize non-specific amplification.

3. Sequencing

• Sequence the prepared libraries on Illumina® platforms, such as Illumina MiSeq™, Illumina NextSeq™, and Illumina NovaSeq™, facilitating high-throughput data generation.

4. Bioinformatics Analysis

• Utilize a cloud-based analysis pipeline to process and quantify on- and off-target edits.
• Interpret results using user-friendly tools, making it accessible to researchers without extensive bioinformatics expertise.

Advantages of our CRISPR off-target validation sequencing

By prioritizing these feature enhancements, our CRISPR off-target validation sequencing ensures researchers can achieve swift, reliable genomic insights, thus facilitating more informed scientific inquiries and discoveries.

Our CRISPR Off-Target Validation Sequencing platform presents several outstanding advantages that position it as an optimal selection for researchers prioritizing precision and reliability in their genomic investigations.

1. Elevated Sensitivity and Specificity: The integration of our proprietary rhAmp PCR technology mitigates off-target amplification, thereby enhancing the precision of target detection and ensuring high-fidelity results.

2. Superior Quantification Capabilities: Our platform delivers unparalleled insertion/deletion (indel) quantification of CRISPR edits, thereby providing researchers with accurate assessments of genome editing proficiency.

3. Advanced Multiplexing Flexibility: Empowering scientists with the capability to multiplex up to 500 targets within a single assay, our solution markedly boosts throughput while simultaneously curtailing associated costs.

4. Efficient Workflow: Our meticulously optimized workflow spans from sample preparation to comprehensive data analysis, offering quick turnaround times that do not sacrifice the integrity or quality of the data produced.

5. Detailed and Comprehensive Reporting: The included analytical tools generate figures and reports of publication quality, which significantly simplify the interpretation, documentation, and dissemination of research findings.

Applications of CRISPR off-target validation sequencing

The CRISPR off-target validation sequencing service is widely applicable across various domains:

• Gene Editing: Ensures the safety of gene-editing therapeutics by verifying off-target edits in clinical applications.
• Agricultural Biotechnology: Validates the specificity of CRISPR edits in genetically modified organisms, enhancing crop traits without unintended effects.
• Basic Research: Supports fundamental genomic studies by providing precise data on the efficiency and specificity of CRISPR techniques in different model systems.
• Pharmaceutical Development: Aids in drug discovery and development by assessing the reliability of gene editing in therapeutic targets.

Genome Editing and Engineering Solutions at CD Genomics

CRISPR Screening Sequencing

CRISPR screening reveals genes linked to drug resistance, making it vital for biomedical research. CD Genomics enhances this process with Next-Generation Sequencing (NGS) for improved efficiency and analysis quality.

CRISPR Validation Sequencing

CRISPR/Cas9 can introduce off-target effects, so validating gene edits is essential. CD Genomics offers precise validation using high-throughput Next-Generation Sequencing (NGS) to ensure reliable results.

CRISPR Off-target Discovery

CRISPR/Cas9 can inadvertently edit unintended genes, leading to off-target effects that may result in harmful mutations. CD Genomics offers high-throughput NGS-based tools to accurately detect off-target sites, ensuring safe and effective gene editing strategies. Our SureGuide OnTarget Platform uses oligonucleotide tags and advanced bioinformatics to pinpoint off-target mutations, enhancing your CRISPR research outcomes.

CRISPR Off-Target Validation Sequencing FAQ

• 1. What is the purpose of CRISPR Off-Target Validation Sequencing?

  • The purpose of CRISPR Off-Target Validation Sequencing is to identify and confirm any unintended edits made during the CRISPR gene editing process, thereby helping researchers detect off-target effects and ensure the precision of their gene editing, ultimately improving both safety and efficacy for therapeutic applications.

• 2. How does the CRISPR off-target validation process work?

  • The process involves collecting DNA samples, constructing custom amplicon libraries targeting both on- and off-target sites, conducting sequencing on Illumina® platforms, and using advanced bioinformatics tools to provide detailed insights into off-target edits.

• 3. What kind of data analysis do you provide?

  • The provided data analysis is easy to understand, highlighting both on-target and off-target editing events, with results delivered through clear, visual reports that simplify the interpretation of the impact of CRISPR edits.

• 4. How can I customize my analysis panel to meet specific needs?

  • Customizing the analysis panel to meet specific needs involves choosing targets based on research objectives and designing a panel tailored to the project, with assistance from our team to ensure the analysis aligns with specific requirements.

• 5. What are the cost benefits of using this technology?

  • The cost benefits of using CRISPR Off-Target Validation Sequencing include achieving high-quality results while reducing experimental costs, as our high-throughput analysis capabilities and optimized experimental designs improve data output and decrease the number of experiments needed, which leads to better cost-efficiency.

• 6. Are there any success stories to demonstrate the effectiveness of this technology?

  • There are several success stories demonstrating how the CRISPR Off-Target Validation Sequencing technology has helped clients achieve significant research outcomes, spanning various fields such as gene therapy and basic research, thus illustrating the broad applications and reliability of the services provided.