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CRISPR/Cas9 PlatformCB provides an off-target prediction and analysis service for CRISPR/Cas9-mediated gene editing. With years of experiences, our scientists are expert in application of CRSIPR/Cas9 system. Our scientist team will provide reliable and professional off-target testing services for our clients. As a leader in gene editing, CRISPR/Cas9 PlatformCB offers trusted analysis results with high quality in a competitive price.
Cas9, which is a nuclease, can be recruited to the specific site by guide RNA and then cleaves DNA. Similarly to other nucleases, Cas9 can cleave off-target DNA targets in the genome at reduced frequencies. For application of Cas9, it is essential to consider ways to minimize the degree of off-target cleavage and to detect the presence of off-target cleavage. Though many tips have been developed to minimize the risk of off-target cleavage, off target mutations is still a major concern about CRISPR/Cas9-mediated genome editing in some instance. These mutations should be carefully monitored, especially when using CRISPR/Cas9 for therapeutic purposes. However, off-target analyses of the CRISPR/Cas9 system have been very challenging, particularly when performed directly in cells. With the wide application of next generation sequence, different sequencing strategies have been developed to identify the off-target mutations, such as whole genome sequencing, GUIDE-seq, Digenome-seq.
CRISPR/Cas9 PlatformCB employs different assays to analyze CRISPR off-target effects, including PCR-based assay and next generation sequencing assays. Methods varies to different situations. The recommendation detection assay is next generation sequencing, including whole-genome sequencing, Guide-seq, Digenome-seq. Next generation sequencing now is widely used for predicting and detecting the off-target mutations by CRISPR/Cas9. Each sequencing assay has own characters and is suitable for different situations. Guide-seq and Digenome-seq are preferred to be used in sgRNA evaluation, while whole-genome sequencing is always applied for measuring the gene-edited cells or animals. Through high-throughput sequencing, it becomes an easy thing to find most of off-target sites. CRISPR/Cas9 PlatformCB provides a complete off-target analysis, ranging from selecting strategy to raw data analysis. Our staff will work closely with you to assist you to detect off-target mutations.
|Methods||Description & Advantages|
PCR-based assay is based on computational predictions and detects the assumptive sites by PCR and sequencing.
|Whole Genome Sequencing|
WGS is an approach to detect the off-target mutations by high throughput sequencing. A reference genome is needed for analysis.
BLESS is an approach to directly detect the DSBs induced by Cas9 nuclease. DSB ends are ligated with biotin, and subsequently collected by using streptavidin.
LAM-HTGTS is a method developed to track translocation events caused by joining between DSBs. It is based on DNA repair by end joining in DSBs.
GUIDE-seq is based on the incorporation of dsODN into DSBs via NHEJ. Its amplification is followed by sequencing.
Digenome-seq is an in vitro method utilizing Cas9's property of cleaving the genome to get an unbiased profile of the entire genome.
SITE-Seq is an in vitro method based on Cas9-cleavage of high molecular weight DNA, followed by enzymatic fragmentation, biotinylated adapter ligation, enrichment and sequencing.
Table 1. Various methods for off-target detection