These days, it hardly seems like we finish writing about one dual CRISPR-transposon system before another exciting new advance emerges! The programmability and targeting power of CRISPR combined with the large sequence capacity of transposons open whole new worlds to explore. The newest arrival on the scene is called evoCAST — an evolved CRISPR-associated transposase (CAST).
Back up a second. What are CASTs?
CASTs occur naturally in bacteria. In the course of evolution, Tn7-like transposons captured and repurposed nuclease-deficient CRISPR-Cas systems to help them spread through bacterial genomes (Peters et al., 2017). Type I CAST systems rely on the Cascade complex minus its nuclease component, Cas3, while Type V-K CAST systems use a nuclease-deficient Cas12k (Gelsinger et al., 2024).
In either type of CAST, the nuclease-deficient CRISPR-Cas system identifies and binds a genomic target using a gRNA, and an affiliated TniQ protein recruits transposition cofactors. The transposon proteins integrate a payload sequence into genomic DNA downstream from the Cas target. Conveniently, the process doesn’t cause double-stranded breaks and can deliver large sequences and even entire genes.
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| Figure 1: Mechanism of CAST systems. A Cas effector (Cascade in Type I systems; Cas12k in Type V-K systems; Cascade shown) combines with a donor template and transposon proteins (Tns) to integrate large sequences into genomic DNA downstream of gRNA-specified sites. Created with BioRender.com. |
CASTs quickly became popular bacterial genome engineering tools, with early reports of 40%–60% efficient integration (Klompe et al., 2019). Type I-F CASTs were found to be particularly useful. But they were barely effective at editing eukaryotic cells — at least, until they met the directed evolution expertise of the Samuel Sternberg and David Liu labs.
Evolving CASTs for new contexts
Natural transposons evolve under contrary selection pressures: high efficiency helps them spread, but also stresses the host. The researchers suspected natural evolution would produce transposons with sub-maximal efficiency, and this barrier could be overcome by directed evolution using bacteriophages.
Researchers moved a gene required for phage propagation to a plasmid that lacked a promoter — only CAST integration of the promoter could turn the gene on. Once phage success was linked to CAST efficiency, evolution took over (with a little mutagenic assistance). Quicker CAST activity allowed certain mutated phages to outcompete their brethren, and the researchers isolated these more efficient CASTs (Witte et al., 2025).
The best evoCAST featured 20 mutations in TnsB, the subunit that catalyzes DNA insertion, along with both evolved and engineered adjustments to the other components. It drove 19% integration in HEK293T cells (more than 500 times better than the precursor it was evolved from!) and was able to integrate DNA payloads of up to 15 kb.
The new horizons of evoCAST
In the emerging world of gene therapy, highly tailored treatments to specific mutations require individualized optimizations, safety testing, and approval. In contrast, a system that can deliver a healthy gene as a single intervention for a variety of loss-of-function mutants could provide a streamlined, one-size-fits-most approach. evoCAST is an important step toward that kind of gene treatment.
The researchers also compared evoCAST to the Liu lab’s own PASSIGE (Prime-Editing-Assisted Site-Specific Integrase Gene Editing), which combines prime editing with a site-specific recombinase to achieve insertion of long sequences. In their experiments, PASSIGE had higher efficiency, but evoCAST required less optimization for new targets and produced a higher-purity product, likely because the integration occurs in a single enzymatic step.
We can’t wait to see where evoCAST takes us on the high seas of gene editing. Bon voyage!
References and Resources
References
Gelsinger, D. R., Vo, P. L. H., Klompe, S. E., Ronda, C., Wang, H. H., & Sternberg, S. H. (2024). Bacterial genome engineering using CRISPR-associated transposases. Nature Protocols, 19(3), 752–790. https://doi.org/10.1038/s41596-023-00927-3
Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S., & Sternberg, S. H. (2019). Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature, 571(7764), 219–225. https://doi.org/10.1038/s41586-019-1323-z
Peters, J. E., Makarova, K. S., Shmakov, S., & Koonin, E. V. (2017). Recruitment of CRISPR-Cas systems by Tn7-like transposons. Proceedings of the National Academy of Sciences of the United States of America, 114(35), E7358–E7366. https://doi.org/10.1073/pnas.1709035114
Witte, I. P., Lampe, G. D., Eitzinger, S., Miller, S. M., Berríos, K. N., McElroy, A. N., King, R. T., Stringham, O. G., Gelsinger, D. R., Vo, P. L. H., Chen, A. T., Tolar, J., Osborn, M. J., Sternberg, S. H., & Liu, D. R. (2025). Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase. Science, 388(6748), eadt5199. https://doi.org/10.1126/science.adt5199
Additional Resources on the Addgene blog
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INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons
- Degrading DNA with Cascade-Cas3
- Typing CRISPR Systems
- Prime Editing: Adding Precision and Flexibility to CRISPR Editing
Additional resources on Addgene.org
- Addgene's CRISPR Guide (Jump to Large Edits)
- CAST Plasmid Collection
Topics: CRISPR, Cas Proteins, Other CRISPR Tools
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