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<p>Despite considerable powers, the application of CRISPR activation (CRISPRa) screens in primary human cells remains a formidable challenge. Here, we develop dCas12f1-SAM, a compact SAM-based transcriptional activation platform, that outperforms existing systems in both immortalized cell lines and primary human T cells and hematopoietic stem/progenitor cells (HSPCs). Using dCas12f1-SAM, we perform a pooled CRISPRa screen targeting 1559 human transcription factors (TFs) in primary human T cells and identify multiple positive regulators of IL-2 expression. We further implement a single-cell CRISPRa screen via our miCROP-seq construct, resolving how these genetic perturbations reshape T cell activation dynamics and drive functionally distinct cellular states. Among the top-ranking genes, we spotlight <i>KLF12</i> and <i>LHX5</i>, whose overexpression significantly improves antigen-specific responses of chimeric antigen receptor T (CAR-T) cells. Collectively, these findings establish dCas12f1-SAM as a robust transcriptional activation tool, highlighting its potential to advance applications in cellular engineering and immunotherapy.</p>

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Engineered dCas12f1-SAM enables robust transcriptional activation and gain-of-function screening in primary human cells

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年5月6日

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  • Jiaxuan Cao,
  • Zhirui Liu,
  • Xinwen Chen,
  • Zilin Lan,
  • Li Liu,
  • Yixin Zhai,
  • Wentian Wang,
  • Chaoyou Xue,
  • Hui Cheng,
  • Yao Yao,
  • Tao Cheng,
  • Shuquan Rao

摘要

Despite considerable powers, the application of CRISPR activation (CRISPRa) screens in primary human cells remains a formidable challenge. Here, we develop dCas12f1-SAM, a compact SAM-based transcriptional activation platform, that outperforms existing systems in both immortalized cell lines and primary human T cells and hematopoietic stem/progenitor cells (HSPCs). Using dCas12f1-SAM, we perform a pooled CRISPRa screen targeting 1559 human transcription factors (TFs) in primary human T cells and identify multiple positive regulators of IL-2 expression. We further implement a single-cell CRISPRa screen via our miCROP-seq construct, resolving how these genetic perturbations reshape T cell activation dynamics and drive functionally distinct cellular states. Among the top-ranking genes, we spotlight KLF12 and LHX5, whose overexpression significantly improves antigen-specific responses of chimeric antigen receptor T (CAR-T) cells. Collectively, these findings establish dCas12f1-SAM as a robust transcriptional activation tool, highlighting its potential to advance applications in cellular engineering and immunotherapy.