CRISPR Off-Target Effects Analysis

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CRISPR Off-Target Effects Analysis    

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.

MethodsDescription & Advantages
PCR-based Assay

PCR-based assay is based on computational predictions and detects the assumptive sites by PCR and sequencing.

Advantages:

  1. 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:

  1. The entire genome is screened for off-target mutations.
  2. 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:

  1. Detect DSBs at nucleotide resolution
  2. Do not depend on proteins that bind to DSB
  3. 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:

  1. Sensitive detection of DSBs within chromsomes
GUIDE-Seq

GUIDE-seq is based on the incorporation of dsODN into DSBs via NHEJ. Its amplification is followed by sequencing.

Advantages:

  1. Generate global specificity landscapes for RGNs in living human cells
  2. Targeted sequencing reduces cost
  3. Highly sensitive
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:

  1. Rely on DNA cleavage rather than binding
  2. Performed in a genomic context and capture sites with a DNA/RNA bulge
  3. Detect off-target effects with a frequency of 0.1% or lower

Table 1. Various methods for off-target detection

For further information, please feel free to contact us at or 1-631-626-9181.

Reference:

  1. Ran FA, et al. (2013) Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 2281-2308
  2. Zhang XH, et al. (2015) ‘Off-target Effects in CRISPR/Cas9-mediated Genome Engineering’, Mol Ther Nucleic Acids 4, e264
  3. Zischewski J, et al. (2016) ‘Detection of on-target and off-target mutation generated by CRISPR/Cas9 and other sequence-specific nucleases’, Biotechnology Advances, 35(1): 95-104
  4. Francisco M, et al. (2016) ‘Biased and unbiased methods for the detection of off-target cleavage by CRISPR/Cas9: an overview’, International Journal of Molecular Sciences,17,1507
  5. Crosetto N, et al. (2013) ‘Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing’, Nature Methods, 10(4): 361-5
  6. Mallin H, et al. (2016) ‘Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology’, Nature Protocols, 11(5): 835-52
  7. Tsai SQ, et al. (2015) ‘GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases’, Nature Biotechnology, 33(2): 187-97
  8. Kim D, et al. (2015) ‘Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells’, Nature Methods, 12(3): 237-43

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