Evidence map›Paper›PMID 41309578›Full record

ArticleNature communications2025

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 and 4 more

Abstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Nature communications, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.

0numbers the graph read from it
0cells of the map it votes in
3citing papers in PubMed
–field-weighted citation impact
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

3 citing papers in PubMed.

  1. Article
  2. Article
  3. Article
4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

14 authors.

Sophia Arana *Department of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.ORCID http://orcid.org/0009-0000-5747-0204
Peter P Du *Department of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.ORCID http://orcid.org/0000-0003-2652-0541
Alun Vaughan-JacksonDepartment of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
Nora EnrightDepartment of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
Kaitlyn SpeesDepartment of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
Raeline ValbuenaDepartment of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
Cesar A GarciaDepartment of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
Thien NguyenDivision of Pediatric Hematology/Oncology, Lucile Packard Children's Hospital, Stanford, CA, USA.ORCID http://orcid.org/0000-0003-2348-0556
Anthony VenidaDepartment of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
Marta SeczynskaDepartment of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
Lacramioara BintuDepartment of Bioengineering, Stanford University, Stanford, CA, USA.ORCID http://orcid.org/0000-0001-5443-6633
Paul J LehnerCambridge Institute for Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge, UK.ORCID http://orcid.org/0000-0001-9383-1054
Laura M ProloDepartment of Neurosurgery, School of Medicine, Stanford University, Stanford, CA, USA.ORCID http://orcid.org/0000-0001-7737-8675
Michael C BassikDepartment of Genetics, School of Medicine, Stanford University, Stanford, CA, USA. bassik@stanford.edu.ORCID http://orcid.org/0000-0001-5185-8427

Funding

Leveraging innovative technologies in basic and clinical cancer researchT32CA009302 · NCI · STANFORD UNIVERSITY · PI LAURA D ATTARDI, Aaron M Newman · 1985 to 2026
$19.1M
Stanford Breast Metastasis Center Administrative CoreU54CA261719 · NCI · STANFORD UNIVERSITY · PI Christina N Curtis · 2021 to 2026
$8.6M
High-throughput development and characterization of compact tools for transcriptional and chromatin perturbationsR01HG011866 · NHGRI · STANFORD UNIVERSITY · PI MICHAEL C BASSIK, Lacramioara Bintu · 2021 to 2026
$6.9M
NCI NIH HHS T32 CA009302NCI NIH HHS U54 CA261719NHGRI NIH HHS R01 HG011866U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI) R01HG011866Wellcome Trust
6 · The paper itself

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.

Indexed as

CRISPR-Associated Protein 9CRISPR-Cas SystemsGene SilencingIntronsTransgenesAnimalsCell LineChromatinGene EditingHEK293 CellsHumansChromatinCRISPR-Associated Protein 9

Identifiers

PMID41309578
PMCPMC12660324

What OpenQuestion holds

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LicenceCC BY-NC-ND
Read underepoch 390

Registered trials

None linked

Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.