Clustered regularly interspaced short palindromic repeats (CRISPR)-based bacterial defense systems can be used for precision genome editing based on single guide RNA (sgRNA) and nuclease Cas9, which can rapidly accelerate plants by precisely modifying genes or genomic regions of interest. Breeding work. In this process, most deletions are created by using two gRNAs, which direct Cas9 to cut out intervening regions of DNA. Therefore, deletions can be detected by PCR using primers flanking the deleted region. Confirming successful gene targeting requires the detection of insertions or deletions introduced by the CRISPR experiment. NGS is a good choice if you have a large number of samples and/or want to look at off-target changes simultaneously. As with any research experiment, validating CRISPR-driven gene editing is critical.

Fig. 1. Application of NGS at different steps of the plant genome editing workflow. (Mushtaq et al., 2021)

Our CRISPR validation sequencing solution

CD Genomics is a trusted partner in the agrigenomics industry, offering comprehensive CRISPR validation sequencing services designed to meet the specific needs of agricultural researchers and breeders. Our state-of-the-art sequencing technology and advanced bioinformatics analyses accurately and efficiently validate CRISPR-induced crop gene editing and can meet the growing demand for CRISPR/Cas9 in animal breeding and crop improvement.

Sanger Sequencing

Sanger sequencing remains the gold standard for CRISPR validation due to its reliability, sensitivity, and accurate identification of introduced mutations. CD Genomics utilizes Sanger sequencing to determine the presence and nature of insertions or deletions (indels) resulting from CRISPR experiments, providing you with highly reliable and detailed information about the specific genetic modifications that have been introduced into the target gene.

Next Generation Sequencing (NGS)

Our NGS provides a powerful platform to support CRISPR validation sequencing in agriculture. NGS has the distinct advantage of eliminating the need to establish clonal cell populations harboring mutations. NGS can even identify rare mutations in subpopulations of cells. Our high-throughput NGS approach provides a comprehensive view of CRISPR-induced modifications, enabling researchers to assess off-target effects and evaluate the overall success of genome editing experiments.

Applications of CRISPR validation sequencing

Our CRISPR validation sequencing solution accurately confirms the presence of desired gene edits and evaluates their impact on target traits, and is designed to help breeders enhance crop resilience, disease resistance, and nutritional traits. NGS validates your CRISPR/Cas9 edits in several ways:

• Whether CRISPR reagents are delivered to your target cells.
• Successful and specific editing of the target gene.
• Expected changes in the expression of the protein encoded by the target gene.

CRISPR validation sequencing workflow

Fig. 2. CD Genomics'CRISPR validation sequencing workflow for agriculture.

Our strengths and features

• Our experts use sophisticated algorithms and analytical processes to interpret sequencing data to provide reliable information for CRISPR validation.
• We understand that different agricultural research projects have unique requirements. CD Genomics provides CRISPR validation sequencing solutions tailored to the specific needs of each project.
• Our team works closely with researchers to design experimental strategies, optimize protocols, and deliver results that meet their goals.
• Our efficient workflows and streamlined processes ensure researchers receive sequencing results within the agreed timeframe.

CD Genomics provides reliable and affordable CRISPR validation sequencing services for agricultural applications using Sanger sequencing and NGS. Targeted NGS-based sequencing provides a cost-effective solution for confirming CRISPR-induced editing by focusing on targeted modified regions. We aim to advance your agricultural project. If you are interested, please feel free to contact us.

Reference

1. Mushtaq, Muntazir, et al. Integrating CRISPR-Cas and next generation sequencing in plant virology. Frontiers in Genetics. 12 (2021): 735489.