Reduced Cas9 transgene silencing by incorporation of intron sequences

Sophia Arana, Peter P. Du, Alun Vaughan-Jackson, Nora Enright, Kaitlyn Spees, Raeline Valbuena, Cesar A. Garcia, Thien Nguyen, Anthony Venida, Marta Seczynska, Lacramioara Bintu, Paul J. Lehner, Laura M. Prolo and Michael C. Bassik ()
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Sophia Arana: Stanford University, Department of Genetics, School of Medicine
Peter P. Du: Stanford University, Department of Genetics, School of Medicine
Alun Vaughan-Jackson: Stanford University, Department of Genetics, School of Medicine
Nora Enright: Stanford University, Department of Genetics, School of Medicine
Kaitlyn Spees: Stanford University, Department of Genetics, School of Medicine
Raeline Valbuena: Stanford University, Department of Genetics, School of Medicine
Cesar A. Garcia: Stanford University, Department of Genetics, School of Medicine
Thien Nguyen: Lucile Packard Children’s Hospital, Division of Pediatric Hematology/Oncology
Anthony Venida: Stanford University, Department of Genetics, School of Medicine
Marta Seczynska: Stanford University, Department of Genetics, School of Medicine
Lacramioara Bintu: Stanford University, Department of Bioengineering
Paul J. Lehner: University of Cambridge, Cambridge Institute for Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus
Laura M. Prolo: Stanford University, Department of Neurosurgery, School of Medicine
Michael C. Bassik: Stanford University, Department of Genetics, School of Medicine

Nature Communications, 2025, vol. 16, issue 1, 1-9

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-65669-0

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