CRISPR Off-Target Effects Analysis    

CRISPR/Cas9 Platform^CB 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 Platform^CB 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, PEM-seq, Digenome-seq.

CRISPR/Cas9 Platform^CB 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, PEM-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. PEM-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 Platform^CB 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	PCR-based assay is based on computational predictions and detects the assumptive sites by PCR and sequencing. Advantages: Easy and cheap
Whole Genome Sequencing	WGS is an approach to detect the off-target mutations by high throughput sequencing. A reference genome is needed for analysis. Advantages: The entire genome is screened for off-target mutations. Unbiased
BLESS	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. Advantages: Detect DSBs at nucleotide resolution Do not depend on proteins that bind to DSB Do not depend on single-strand DNA
LAM-HTGTS	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. Advantages: Sensitive detection of DSBs within chromsomes
Digenome-Seq	Digenome-seq is an in vitro method utilizing Cas9's property of cleaving the genome to get an unbiased profile of the entire genome. Advantages: Rely on DNA cleavage rather than binding Performed in a genomic context and capture sites with a DNA/RNA bulge Detect off-target effects with a frequency of 0.1% or lower

Table 1. Various methods for off-target detection

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