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SITE-Seq    

Creative Biogene CRISPR/Cas9 PlatformCB provides SITE-Seq service to assess genome-wide off-target effects of CRISPR/Cas9. We utilize advanced NGS technology to help you identify actual off-target sites, as well as precisely calculate on/off-target frequency. Our team members have experience in both high-throughput sequencing and genome editing, enabling extensive support to your research.

Advances in genome editing technology, in particular the CRISPR/Cas9, have revolutionized gene editing with promise for advances in basic science, biotechnology, and biomedical research. With the simplicity of modern gene editing technologies come problematic off target interactions which can lead to unintended effects in research and clinical applications. Therefore, high-sensitive genome-wide methods are required to fully understand the genomic locations and frequency of nuclease-induced DSB activity.

SITE-Seq is an in vitro and biochemical method that uses the selective enrichment and identification of tagged genomic DNA ends by sequencing to identify Cas9 cleavage sites in purified genomic DNA. Since SITE-Seq is a biochemical assay, genomic DNA can be digested with a range of sgRNP concentrations, from limiting to saturating, thus permitting the recovery of both high- and low-cleavage-sensitivity off-target sites. This can then be used to guide careful and comprehensive examination of possible off-target sites in cells, measuring both mutation frequency and functional cellular consequence. Moreover, SITE-Seq is a high-throughput guide selection tool, which produces highly enriched sequencing libraries and enables specificity profiling with minimal read depth.

Workflow of SITE-Seq

  • Extraction and purification of high-molecular weight genomic DNA
  • Execution of Cas9 Ribonucleoprotein (RNP) cleavage
  • Fragmentation, adapter ligation, and affinity purification to enrich for Cas9 cleaved fragments
  • Amplification and indexing of SITE-Seq libraries for high-throughput sequencing

SITE-SeqFigure 1. Schematic overview of SITE-Seq

Comparing to GUIDE-Seq and HTGTS, SITE-Seq is not reliant on cellular events such as DNA repair, which will likely be influenced by Cas9 delivery method, cell type and target site. SITE-Seq based analysis covers many hundreds of potential off-targets, including previously identified off-target sites and additional potential off-target sites. Moreover, SITE-Seq combines with cell-based validation, is expected to a robust pipeline for selecting sgRNPs and delivery methods with maximized activity and specificity.

Talent scientists of Creative Biogene CRISPR/Cas9 PlatformCB assists you in genome-wide off-target effects analysis using SITE-Seq. Our complete service covers from sample preparation, sequencing to data analysis. Once project finishes, reliable report and raw data will be delivered to customers. For more information, please contact us at or 1-631-626-9181. We are waiting for your interest projects.

References

  1. Cameron, P, et al. (2017). 'Mapping the genomic landscape of crispr-cas9 cleavage'. Nature Methods.
  2. Cameron, P, et al. (2017). 'Site-seq: a genome-wide method to measure cas9 cleavage'. Protocol Exchange
  3. Kim, D, (2019). 'Evaluating and enhancing target specificity of gene-editing nucleases and deaminases'. Annual Review of Biochemistry, 88(1).
For research use only. Not intended for any clinical use.

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