2. Academic Validation
  3. Reduced Cas9 transgene silencing by incorporation of intron sequences

Reduced Cas9 transgene silencing by incorporation of intron sequences

  • Nat Commun. 2025 Nov 27;16(1):10656. doi: 10.1038/s41467-025-65669-0.
Sophia Arana # 1 2,  Peter P Du # 1 3,  Alun Vaughan-Jackson 1 4,  Nora Enright 1,  Kaitlyn Spees 1,  Raeline Valbuena 1,  Cesar A Garcia 1,  Thien Nguyen 5,  Anthony Venida 1,  Marta Seczynska 1,  Lacramioara Bintu 6,  Paul J Lehner 7,  Laura M Prolo 2,  Michael C Bassik 8
Affiliations

Affiliations

  • 1 Department of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
  • 2 Department of Neurosurgery, School of Medicine, Stanford University, Stanford, CA, USA.
  • 3 Cancer Biology Program, School of Medicine, Stanford University, Stanford, CA, USA.
  • 4 Chan Zuckerberg Biohub, San Francisco, CA, USA.
  • 5 Division of Pediatric Hematology/Oncology, Lucile Packard Children's Hospital, Stanford, CA, USA.
  • 6 Department of Bioengineering, Stanford University, Stanford, CA, USA.
  • 7 Cambridge Institute for Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge, UK.
  • 8 Department of Genetics, School of Medicine, Stanford University, Stanford, CA, USA. [email protected].
  • # Contributed equally.
PMID: 41309578 DOI: 10.1038/s41467-025-65669-0
Abstract

Silencing remains a significant challenge for exogenous gene expression, limiting both the penetrance and expressivity of transgenes. In particular, silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here, we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably, the incorporation of an intron into a CRISPRa construct results in reduced silencing, increased expression levels, and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition, we find that introns can work synergistically with chromatin opening elements to further mitigate silencing, suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes.

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