ArticleMolecular therapy : the journal of the American Society of Gene Therapy2025
Orthogonal transcriptional modulation and gene editing using multiple CRISPR-Cas systems.
Article in Molecular therapy : the journal of the American Society of Gene Therapy, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- The application of CRISPR gene-editing technology in influenza prevention and control.Frontiers in genome editing · 2026Review
- Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of allogeneic CAR-T cells.Molecular therapy : the journal of the American Society of Gene Therapy · 2025Article
- Cytotoxicity of activator expression in CRISPR-based transcriptional activation systems.Nature communications · 2025Article
- Programmable genome engineering and gene modifications for plant biodesign.Plant communications · 2025Review
- Three's company: Simultaneous trimodal genome engineering using orthologous Cas proteins.Molecular therapy : the journal of the American Society of Gene Therapy · 2025Article
Corrections and comments
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
CRISPR-Cas-based transcriptional activation (CRISPRa) and interference (CRISPRi) enable transient programmable gene regulation by recruitment or fusion of transcriptional regulators to nuclease-deficient Cas (dCas). Here, we expand on the emerging area of transcriptional engineering and RNA delivery by benchmarking combinations of RNA-delivered dCas and transcriptional modulators. We utilize dCas9 from Staphylococcus aureus and Streptococcus pyogenes for orthogonal transcriptional modulation to upregulate one set of genes while downregulating another. We also establish trimodal genetic engineering by combining orthogonal transcriptional regulation with gene knockout by Cas12a (Acidaminococcus; AsCas12a) ribonucleoprotein delivery. To simplify trimodal engineering, we explore optimal parameters for implementing truncated single guide RNAs (sgRNAs) to make use of SpCas9 for knockout and CRISPRa. We find the Cas9 protein/sgRNA ratio to be crucial for avoiding sgRNA cross-complexation and for balancing knockout and activation efficiencies. We demonstrate high efficiencies of trimodal genetic engineering in primary human T cells while preserving basic T cell health and functionality. This study highlights the versatility and potential of complex genetic engineering using multiple CRISPR-Cas systems in a simple one-step process yielding transient transcriptome modulation and permanent DNA changes. We believe such elaborate engineering can be implemented in regenerative medicine and therapies to facilitate more sophisticated treatments.
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Registered trials
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.