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Off-target Analysis for CRISPR/Cas9

Since CRISPR/Cas9 system was first applied for genome engineering, it becomes one of the most popular tools in the worldwide. Considering its easy-to-use and simple-to-design, researchers are prone to CRISPR/Cas9 technique for genome editing in most cases rather than others. However, off-target cleavage is a major concern of CRISPR/Cas9 for genome editing in some instance. For clinical application, identification of even low-frequency alterations is critically important when CRISPR RNA-guided nucleases are involved in therapeutic strategies. To date, various assays have been developed to figure this question, including biased methods and unbiased methods.

1. PCR-based Assay

The PCR based assay is based computational predictions and then detects the assumptive sites by PCR and sequencing. Since the potential off-target sites is predicted following the rule of the similarity of sequence, this assay is biased and may miss some off-target sites. Therefore, this method is rarely used alone though it is easy and cheap, and always applied together with other assays for verification.

2. Whole Genome Sequencing

Whole genome sequencing is an unbiased approach to detect the off-target mutations by high throughput sequencing technique. By comparing the editing genome with reference genome, WGS can screen the off-target mutations in genome-wide. Not only small indels and SNPs, but also structural variants such as inversions, rearrangements, duplications and major deletions, can be identified by WGS.

3. GUIDE-Seq

GUIDE-Seq, as a cell-based unbiased assay, screens the entire genome for DNA double-strand breaks introduced by CRISPR RNA-guided nucleases and potentially other nucleases. Sequence-specific double stranded oligodeoxynucleotides (dsODN) are integrated into double strand breaks via NHEJ. Then mapping the insertion sites by NGS. GUIDE-seq is adapted to identify all off-target sites that occur with frequencies above ~0.1% in different cells.

Off-target Analysis for CRISPR/Cas9 Fig 1. GUIDE-Seq2


CIRCLE-Seq, an in vitro biochemical method for finding off-targets, enables people to detect off-target mutations occur with frequencies below ~0.1%. This method is based on NGS, but does not required reference genome sequence. Additionally, CRICLE-seq can be applied for identification of off-target mutations associated with cell-type-specific single-nucleotide polymorphisms. CIRCLE-seq provides an accessible, rapid and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9.

Off-target Analysis for CRISPR/Cas9 Fig 2. CIRCLE-Seq3

Creative Biogene employs various methods for off-target analysis of CRISPR/Cas9, including WGS, GUIDE-Seq and CIRCLE-Seq. Based on excellent CRISPR/Cas9 platform, all tests are performed by trained staff under the guidance of experienced scientists. Reliable report and raw data will delivered to customers once projects finish.


  1. Zischewski J, et al. ‘Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases’, Biotechnology Advances, 2017
  2. 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
  3. Tsai SQ, et al. (2017) ‘CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets’, Nature Methods, 14(6)
For research use only. Not intended for any clinical use.

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