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Efficiency and specificity are two problems that cannot be evaded with gene editing tools. Precise detection of off-target activities is an essential step in evaluating the practicability of CRISPR system. Thus, assays for the sensitive detection and analysis of off-target editing are critical. Creative Biogene has established a comprehensive analytical platform for detecting off-target effects. The platform includes a variety of advanced assays to provide customers with comprehensive and accurate CRISPR/Cas9 off-target effect detection and analysis services.
The CRISPR-Cas system allows targeted editing of genes of interest in various organisms, from bacteria to humans, and has versatile applications in vivo. However, when the CRISPR system recognizes sequences similar to the target sequence, deleterious off-target mutations can occur, especially in mammals given their large genomes, which can lead to malfunctions. Cleavage at off-target sites can lead to chromosomal rearrangements, including deletions, insertions, and translocations, which may result in the disruption of normal gene expression and the activation of oncogenes. Various CRISPR effectors such as Cas9 and Cas12a (Cpf1) suffer from off-targeting due to guide (g)RNA properties. Consistently, it is necessary to comprehensively test and analyze the off-target risk of CRISPR to improve the safety of CRISPR technology and screen the safest sgRNA sequences to meet the needs of researchers.
Fig. 1 Effects of off-target mutation on animal and plant phenotype. (Naeem, M.,et al, 2020)
Creative Biogene provides comprehensive and accurate CRISPR off-target effect analysis services. Our experienced scientists can effectively analyze the potential off-target sites of sgRNA, reduce the probability of off-target site editing occurring, and help our clients assess the off-target risk in a timely manner. In addition, our services can also be used for early mass screening in preclinical studies. Through rapid screening of a large number of sgRNAs, we can help customers find sgRNAs with lower off-target risk.
Creative Biogene uses different assays to analyze CRISPR off-target effects, including in vitro and in vivo detection. Here, we describe current analysis platforms at Creative Biogene to detect off-target effects including genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-Seq), high-throughput genomic translocation sequencing (HTGTS), breaks labeling, enrichments on streptavidin and next-generation sequencing (BLESS), and in vitro nuclease-digested genome sequencing (Digenome-seq). Each of these methods has its own characteristics and is suitable for different situations. Our experts will work closely with you to provide you with customized assays and analysis strategies.
| Methods | Description |
|---|---|
| PCR-based Assay | Predict off-target sites using software, PCR amplify the predicted off-target sites, and detect the off-target status by sequencing or zymography. Easy to operate and cost-effective. |
| Whole Genome Sequencing | A method for detecting off-target mutations by high-throughput sequencing. Appropriate reference genomes need to be selected to filter background mutations, potential modification sites containing PAM motifs are obtained by data comparison analysis, and further gene amplification is performed to verify the mutations. |
| BLESS | BLESS is the latest method for the genome-wide mapping of DSBs introduced by Cpf1, Cas9 and nucleases. This technique offers the advantage of being more versatile, sensitive, and quantitative than other DSB mapping methodologies. |
| GUIDE-Seq | GUIDE-seq based on the incorporation of oligodeoxynucleotides (dsODN) in DSBs followed by the NHEJ DNA repair pathway and the integrated dsODN later amplified to detect and quantify the off-target effects. |
| LAM-HTGTS | LAM-HTGTS is a robust, sensitive and unbiased in vivo off-target detection method which can detect the chromosomal translocations in cultured mammalian cells. The advantages of LAM-HTGTS include the ability of the method to identify all known classes of genome-wide recurrent DSBs induced by on/off-target activity of engineered nucleases. |
| Digenome-Seq | Digenome-seq is a genome-wide assay for the in vitro detection and quantification of off-target effects of Cas9 and other nucleases. The sensitivity of Digenome-seq is 0.1% or lower. |
| CIRCLE-seq | CIRCLE-Seq is an in vitro whole genome assay that can detect 94% off-target sites. The assay process involves breaking and cyclizing gDNA, incubating the cyclized DNA with CRISPR/RNP, and further off-target detection by NGS. |
Creative Biogene combines our expertise with state-of-the-art assay platforms to accurately analyze off-target sites on a genome-wide scale, advancing the use of CRISPR/Cas9 technology in therapeutic drug development and clinical research. Contact us to learn more about our services.
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