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Objective <p>The efficiency of CRISPR interference (CRISPRi) depends on functional dCas9 activity, yet practical and reproducible validation of dCas9-expressing cell lines remains limited. Here, we describe a simple and reproducible assay to assess dCas9 functionality using a single sgRNA targeting the nonessential and ubiquitously expressed surface protein CD81.</p> Results <p>We evaluated this approach in multiple hematological and solid tumor cell lines expressing the dCas9-KRAB-MeCP2 repressor complex. In all tested models, CD81 targeting resulted in a consistent reduction of surface protein levels, quantified by flow cytometry. This assay provides a rapid and quantitative functional readout of dCas9 activity without the need for reporter constructs or transcriptional assays. The CD81-targeting sgRNA and validated cell lines are made available to support reproducibility and technical standardization in CRISPRi experiments. This strategy can be readily implemented in any laboratory using CRISPRi-based approaches.</p>

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Validation of a CD81-based flow cytometry assay to assess dCas9 silencing activity

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月8日

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  • Iris Manosalva,
  • Megan Charles-Alfred,
  • Magali Torres,
  • Joaquin Felipe Roca Paixao,
  • Salvatore Spicuglia

摘要

Objective

The efficiency of CRISPR interference (CRISPRi) depends on functional dCas9 activity, yet practical and reproducible validation of dCas9-expressing cell lines remains limited. Here, we describe a simple and reproducible assay to assess dCas9 functionality using a single sgRNA targeting the nonessential and ubiquitously expressed surface protein CD81.

Results

We evaluated this approach in multiple hematological and solid tumor cell lines expressing the dCas9-KRAB-MeCP2 repressor complex. In all tested models, CD81 targeting resulted in a consistent reduction of surface protein levels, quantified by flow cytometry. This assay provides a rapid and quantitative functional readout of dCas9 activity without the need for reporter constructs or transcriptional assays. The CD81-targeting sgRNA and validated cell lines are made available to support reproducibility and technical standardization in CRISPRi experiments. This strategy can be readily implemented in any laboratory using CRISPRi-based approaches.