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Coloring target and off-target effects of genetically modified nucleases by blue & white colony assays

Xumeng Chen1, Li Xiao2, Qian Wang2

  • 1National Engineering Research Center of Personalized Diagnostic and Therapeutic Technology, Hunan University of Chinese Medicine, Changsha, 410208, China.

Biotechnology Letters
|November 7, 2025
PubMed

Insights

New chromogenic assays offer sensitive evaluation of CRISPR/Cas9 gene editing, crucial for advancing human gene therapy by detecting off-target effects. These assays improve safety assessments for gene editing technologies.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Gene Therapy

Background:

  • Accurate assessment of gene editing nucleases' off-target effects is vital for safe human gene therapy.
  • CRISPR/Cas9 technology holds promise for therapeutic applications but requires rigorous safety evaluations.

Purpose of the Study:

  • To develop sensitive chromogenic assays for evaluating gene editing activities, specifically targeting CRISPR/Cas9.
  • To establish robust methods for both qualitative and quantitative assessment of on-target and off-target effects.

Main Methods:

  • Utilized beta-galactosidase alpha complementation for assay development.
  • Employed color alteration in E.coli colonies for evaluating CRISPR/Cas9 gene editing outcomes.
  • Designed assays to detect off-target effects with mismatched bases in target sites and guide RNAs (gRNAs), including mismatched PAM sites.

Main Results:

  • Successfully established chromogenic assays for sensitive evaluation of CRISPR/Cas9 gene editing.
  • Demonstrated the capability to profile off-target effects with high sensitivity, even with single base mismatches.
  • Enabled identification of gene editing effects at off-target sites with one base mismatched PAM sites.

Conclusions:

  • The developed chromogenic assays provide a sensitive, efficient, and versatile tool for evaluating CRISPR/Cas9 gene editing.
  • These assays are critical for enhancing the safety profile of gene editing technologies in therapeutic development.
  • The methodology facilitates comprehensive profiling of gene editing specificity, essential for clinical translation.
Keywords:
Blue & white assaysCRISPR/Cas9Off-target effectsPAM sites

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