Target Region Sequencing

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Target Region Sequencing    

Creative Biogene CRISPR/Cas9 PlatformCB has established a cost-effective, high-throughput strategy for identifying CRISPR/Cas-induced on/off-target mutations via deep sequencing of PCR amplicons. Based on years of experience in genome editing and off-target effects analysis, we offer one-stop solution to accurately detecting mutations induced by sequence-specific nucleases. Our talent scientists will work with you closely to assist you in achieving your research goal.

CRISPR system now holds great promise in diverse fields such as animal disease modeling, material science, genetically modified plant technology, biofuel technology, gene therapy, and drug development. However, off-target effect is still one major concern when applying CRISPR system to biomedical and clinical application. Although a series of genome-wide, in vitro/vivo and biased/unbiased methods are developed to detect off-target effects caused by engineered nuclease, such as TALEN, Cas9, Cpf1 and so on. Detecting off-target sites in a highly sensitive and comprehensive manner remains a key challenge in the field of gene editing.

Target region sequencing is a highly targeted approach that enables researchers to analyze genetic variation in specific genomic regions. The ultra-deep sequencing of PCR products (amplicons) allows efficient variant identification and characterization. It in combination with genome-wide off-target detection methods (SITE-seq, GUIDE-seq, WGS) allows to determine the true on-target/off-target mutations of genome editing experiments with high sensitivity and in a non-biased manner.

Target Region SequencingFigure 1. Workflow of target region sequencing

Workflow of target region sequencing

  • CRISPR plasmids/RNP transfection.
  • Genomic DNA isolation.
  • Determine the potential off-target sites using genome-wide off-target detection method.
  • First step PCR with site-specific primer to amplify the target region.
  • Second step PCR for NGS adaptors addition.
  • Next-generation sequencing
  • Data analysis

CRISPR/Cas9 PlatformCB provides a high-throughput strategy for identifying CRISRP/Cas-induced on/off target mutations via deep sequencing of PCR amplicons (target region). We offer a complete service from sample treatment, primer design to amplicon sequencing. We can handle PCR amplicons of any sizes using different sequencing configuration. All detailed information about on/off target mutations will deliver to you, including confirmation of knockout/knockin alleles, assessment of sgRNAs cutting efficiency, homozygous and heterozygous identification, validation of potential off-target sites. Our experienced technical team will offer an entirely customized solution according specific project. For more information, please contact us at or 1-631-626-9181. We are waiting for your interest projects.

Reference

  1. Anderson, K. R, et al. (2018). Crispr off-target analysis in genetically engineered rats and mice. Nature Methods.
For research use only. Not intended for any clinical use.

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