CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) genome editing enables precise, targeted genetic modification of plants. To date, the genomes of several crops, including rice, tomato, corn, wheat, soybean, barley, potato, sorghum, apple, grapefruit, and orange, have been edited using CRISPR-Cas-based genome editing technology. The CRISPR system cuts not only at the target site, but also at unintended sites with similar sequences. As a result, unintended off-target edits may occur, which may pose risks when present in gene-edited food crops. Current methods include targeted sequencing, exome sequencing, WGS (whole genome sequencing), BLESS (direct in situ fragmentation labeling, streptavidin enrichment, second-generation sequencing), GUIDE-seq (whole genome, no Biased identification of DSB), LAM-HTGTS (linear amplification-mediated high-throughput whole-genome translocation sequencing) and Digenome-seq (in vitro Cas9 digested whole-genome sequencing) are used to detect off-target mutations. Among them, targeted sequencing and WGS are currently widely used in plant off-target analysis.

Fig. 1. Schematic diagram of on-target and off-target analysis by whole-genome sequencing. (Wang et al., 2021)

Our CRISPR off-target sequencing solution

At CD Genomics, we understand the importance of accurate and comprehensive off-target analysis in agricultural applications of CRISPR technology. We specialize in high-quality agricultural genomic services, providing state-of-the-art CRISPR off-target sequencing solutions tailored to the agricultural industry. We ensure accurate detection of CRISPR off-target cut sites for you.

Our CRISPR off-target sequencing service combines WGS with advanced bioinformatics analysis to comprehensively identify potential off-target sites and detect any unintended mutations, including single nucleotide polymorphisms (SNPs), insertions/deletions (indels), and structural variations, which have been widely used to detect off-target mutations in plants such as Arabidopsis thaliana, rice, tomato, and cotton.

Genome-wide NGS to identify CRISPR off-target

Our unbiased WGS off-target assays are a reliable option for off-target analysis during the development of your gene-edited plants, and they allow the detection of off-target changes in genomic regions (coding and non-coding) that cannot be recognized by computer off-target prediction. We aim to help breeders accelerate animal breeding and crop improvement. Our NGS platform provides you with the following information:

• Discover mismatched gRNAs and off-target sites
• Discover types of off-target modifications (insertions or deletions and their size, nucleotide substitutions)
• Discover the location of off-target modifications (Coding or non-coding region, if specified, gene name and its accession number)

NGS-based CRISPR off-target analysis methods for agriculture

Although targeted sequencing of target editing sites can be performed without knowledge of off-target effects, it is often critical to understand the off-target effects in an experiment. Therefore, potential off-target effect sites must first be identified before off-target editing can be properly evaluated. CD Genomics offers several NGS-based whole-genome sequencing to detect off-target mutations. Our approach takes into account nucleotide mismatches, protospacer adjacent motif (PAM) sequences, and potential binding and cleavage efficiencies of single guide RNAs (sgRNAs) and distinguishes true off-target mutations from genetic variation and background noise.

We'll choose the method that's best for your project, depending on your experimental system and specifically the Cas enzyme you're using for genome editing.

• Cell-based (live cells) genome-wide assays
• In vivo genome-wide assays

In addition, we offer the following strategies to reduce off-target effects to advance your agricultural projects:

Cas9 improvement

• SpCas9 variants
• Cas9 orthologous

gRNA improvement

• gRNA length adjustment
• Chemical modifications

Delivery methods improvement

• Application in cell culture
• Application in vivo

CD Genomics is committed to providing agricultural researchers with reliable CRISPR off-target sequencing services to detect off-target sites and off-target modification details in CRISPR-Cas gene-edited plants and animals. By harnessing the power of NGS to reduce the risks associated with off-target mutations, we help you develop sustainable and improved crop varieties. Our expertise, cutting-edge technology, customized solutions, and comprehensive reports make us a trusted partner in the field of agrigenomics research. If you are interested, please feel free to contact us.

Reference

1. Wang, Xianhang, et al. Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine. Horticulture research. 8 (2021).