CRISPR/Cas9 Off-Target Effect Analysis

Since the CRISPR/Cas9 system was first described, it has been used in gene editing in laboratories around the world. Off-target phenomenon of CRISPR/Cas9 has been reported in many Drosophila laboratories, which seriously affects functional analysis after gene editing. In short, Cas9 binds to unintended genomic sites for cleavage, termed as off-target effects. The efficiency and success of editing genes may be affected by several factors. Furthermore, off-target effects could lead to lethal genetic mutations that cause loss of gene function, ultimately confusion and variation in Drosophila or other animals. To this end, we offer the Cas9 off-target effect analysis supporting a more complete and effective gene-editing service.

CD BioSciences as an expert at CRISPR/Cas9 genomic editing in Drosophila, uses leading edge technology to assure your every step of precise and high-efficient editing operations. Our unique platform provides different assessment packages ranging from individual detection to whole genome analysis, from in vitro to in vivo assays that are personalized to the needs of clients. Our professional scientists’ team will put in complete analysis report and professional suggestions in the way of improvement, to help you find and solve problems quickly and effectively.

CRISPR/Cas9 Off-Target Effect Analysis

Our Service

Off-target effect remains a major concern for CRISPR/Cas9-mediated genome editing. However, off-target analysis of CRISPR/Cas9 systems is very challenging, especially when performed directly in vivo. Choosing a suitable off-target assay is more critical and tractable than improving the CRISPR delivery system. Recommended assays are second-generation sequencing, including whole-genome sequencing, Digenome-seq, etc. Each sequencing assay has its own characteristics and is suitable for different situations. E.g., Digenome-seq are preferentially used for sgRNA assessment, while whole-genome sequencing is always used to measure gene edited cells or Drosophila.

Methods Description Advantages Limitations
PCR-based Assay Based on computational predictions, detecting single or serval sites mutation. Cheap and quick Poor sensitivity; no larger rearrangements detection.
Whole Genome Sequencing Widely used in vitro detection of unbiased target Detect miss sites at indels frequencies of 0.1% or less Reference genome needed; challenges with serval sgRNA.
Digenome-seq in vitro Cas9-digested whole-genome sequencing Robust, sensitive and widely used; cost-effective No large-scale screening of gRNA

Table 1. Methods for Drosophila off-target detection

How Does Our CRISPR/Cas9 Off-Target Analysis Service Do?

Although the targeting specificity of Cas9 is tightly controlled by the 20 nt sequence of sgRNA and PAM adjacent to the target sequence in the genome, the complex chromosomal structure and microenvironment of Drosophila in vivo may lead to potential off-target effect. We have a well-established and efficient CRISPR off-target effect analysis and prediction platform, as well as Drosophila database, to find the core problem and provide off-target improvement solutions for customers.

  • sgRNA
    The sgRNA sequence is considered to be a key factor in improving cleavage efficiency and targeting specificity. To reduce off-target effects, we have a complete optimization strategy including GC content, sgRNA length and chemical modifications.
  • PAM
    Drosophila chromatin accessibility is an important determinant of binding efficiency in vivo. As a result, the binding frequency of PAM sequence at each site is different. Our sequencing and prediction platform helps you find the most suitable PAM loci.
  • Cas9 Protein and other factors
    We have built a bioinformatics analysis platform for Drosophila Cas9 protein activity from complex experimental data as well as a biochemical analysis platform. Whether injecting Cas9 protein or mRNA, or establishing a Cas9 transgenic strain, we will carefully analyze the effect of in vivo environment on Cas9 protein activity for our clients to analyze the cause of off-target and reduce off-target effects.

CD BioSciences strives to deliver CRISPR/Cas9 editing technologies and cost-effective off-target analysis that deepen exploration of Genetics and Cell Biology in Drosophila research. We promise to assistant you a complete off-target effect service covering sample preparation, selection of optimal detection, sequence analysis to follow-up scheme optimization relying on analysis reports. If you have any questions or requirements about our services, please feel free to contact us.


  1. Gratz SJ, et al. (2015). Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9. Methods Mol Biol. 1311, 335–348.
  2. Naeem M, et al. (2020). Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing. Cells. 9(7), 1608.

For research use only. Not intended for any clinical use.

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