2026

Background#

Delivery of protein–RNA complexes#

Protein–RNA complexes can combine a protein's catalytic activity with an RNA's sequence-specific recognition. CRISPR–Cas9 is a clear example: the guide RNA specifies a DNA target, while Cas9 cleaves it. Changing the guide sequence can retarget the complex without redesigning the protein.1

Delivery must preserve both components and bring them into the same cell and intracellular compartment. Uptake across the cell membrane and escape from endosomes are major barriers. Formulation conditions also matter: Wei and co-workers found that acidic conditions used for conventional lipid nanoparticle formulation destabilised Cas9 ribonucleoproteins, and developed a modified formulation that retained activity and enabled tissue-specific editing in mice.2

A shared carrier is one way to coordinate delivery, but protein and RNA do not necessarily require separate vehicles. Wang and co-workers delivered Cas9–guide RNA complexes using bioreducible lipid nanoparticles, and Wei and co-workers extended lipid-based ribonucleoprotein delivery to systemic administration. Protein nanocages offer an additional, genetically programmable platform for combining protein and RNA cargo.3212

Encapsulins#

Encapsulins are prokaryotic protein nanocompartments. Well-characterised icosahedral examples include T=1 shells with 60 subunits, T=3 shells with 180 subunits, and T=4 shells with 240 subunits. Sutter and co-workers established the structural basis of enzyme encapsulation in a T=1 cage; Giessen and co-workers subsequently characterised the approximately 42 nm T=4 shell of Quasibacillus thermotolerans, QtEncapsulin, as part of an iron-storage system.45

In the Family 1 encapsulins used here, a short cargo-loading peptide (CLP) on the cargo binds a site on the shell's inner surface. This genetically encoded recognition can be transferred to non-native proteins. Cassidy-Amstutz and co-workers identified a minimal loading tag, while Altenburg, Rollins, Silver and Giessen examined how targeting-peptide sequence, length and cargo properties influence loading.67

The shell exterior and interior can be engineered for different purposes. Examples include simultaneous antigen display and protein loading for vaccine research, and the Giessen lab's engineering of encapsulins for concurrent RNA and protein packaging.812

Permeability of QtEncapsulin#

Established loading strategies include co-expression of cargo and shell, and controlled disassembly followed by reassembly. These approaches have different requirements rather than a universal need for harsh treatment: Jones, Cristie-David, Andreas and Giessen engineered QtEncapsulin to undergo reversible disassembly under mild, controllable conditions.69

Kwon, Andreas, Jones and Giessen demonstrated that pre-assembled QtEncapsulin can instead internalise CLP-tagged proteins in a single mixing step. Their experiments included cargoes from 14 to 482 kDa, much larger than the shell's static pores. Their structural observations support local shell flexibility and transient openings as a route for entry. This is a proposed loading mechanism, not evidence that a large protein passes through an unchanged pore.10

The same study developed a modified QtEnc-based nanocarrier with pH-responsive cargo detachment and an endosomal-escape module, and demonstrated cytosolic protein delivery in HeLa cells. These results establish prior delivery work by the Giessen lab; our bacterial selection and proposed protein–RNA cargo design address different experimental questions.10

Rational pore engineering. Kwon, Andreas and Giessen engineered the encapsulin of Myxococcus xanthus to improve molecular transport and access of substrates to encapsulated enzymes. This work concerns a related encapsulin, rather than QtEncapsulin, and shows how pore design can improve nanoreactor performance.11

Concurrent RNA and protein packaging. Kwon and Giessen modified encapsulin shells with nucleic-acid-binding peptides while preserving native protein loading. Their 2022 study demonstrated size-selective RNA packaging, packaging of multiple functional RNAs, and concurrent RNA and protein encapsulation in living cells. This work provides a direct precedent for our protein–RNA co-packaging strategy.12

Directed evolution of QtEncapsulin. Siddiquee, Lie, Szyszka, Loustau, Andreas, Giessen and Lau used a chloramphenicol selection in which encapsulation protects tagged chloramphenicol acetyltransferase from degradation. Their two-plasmid design pairs a variable shell gene with an invariant wild-type copy, compensating for both metabolic burden and assembly fitness. This enables selection of functional homomeric variants and variants that require mixed assemblies with wild-type subunits. Their results also identify variants that disrupt assembly at higher mutant-to-wild-type ratios, highlighting the need to test shell variants in their intended genetic context.13

Evolution of an RNA-packaging capsid. Terasaka, Azuma and Hilvert engineered and evolved lumazine-synthase cages to package their own RNA. Tetter and co-workers subsequently evolved this platform under increasing nuclease challenge, improving packaging and protection of full-length RNA and producing a virus-like architecture. These cages are derived from a bacterial enzyme, rather than an encapsulin shell.1415

Open problems#

Cargo recognition. Native Family 1 encapsulins use protein targeting peptides; engineered RNA recognition and protein–RNA co-packaging have already been demonstrated. Our question is whether QtEncapsulin can combine its native protein-loading route with engineered λN–boxB RNA recognition for the selected dCas9–guide RNA cargo. The design builds on the encapsulin work above and on the characterised λN–boxB interaction.41216

Measurement. Prior studies have measured purified cargo-loaded particles, and Siddiquee and co-workers established a survival-based encapsulin selection. Our proposed circuit uses a different readout: dCas9 and its guide repress a kanamycin-resistance gene, and sequestration of either component should relieve repression. CRISPR interference is established, but growth rescue alone would not demonstrate that both components occupy the same cage. Independent assembly and cargo-loading assays remain necessary.71317

Throughput. Continuous diversification can reduce repeated cycles of library construction and transformation, but each platform needs a suitable selection. PACE couples activity to phage propagation; OrthoRep uses an orthogonal, error-prone replication system in yeast; T7-ORACLE uses an engineered T7 replisome in E. coli. These are distinct approaches to sustaining mutation and selection in vivo.181920

MutaT7, developed by Moore, Papa and Shoulders, fuses a cytidine deaminase to T7 RNA polymerase to favour mutations downstream of a T7 promoter. Terminator arrays can delimit the targeted region, although targeting should not be interpreted as complete absence of off-target mutations. We propose using this system to diversify the shell cassette alongside our growth selection; the continuous evolution campaign remains planned.21

The design, mechanism, and results pages distinguish our proposed circuit from the results obtained so far. Full citations are collected below and on the references page.

  1. M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna & E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”. Science 337, 816–821 (2012). doi:10.1126/science.1225829. ↩

  2. T. Wei, Q. Cheng, Y.-L. Min, E.N. Olson & D.J. Siegwart, “Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing”. Nature Communications 11, 3232 (2020). doi:10.1038/s41467-020-17029-3. ↩↩

  3. M. Wang, J.A. Zuris, F. Meng, H. Rees, S. Sun, P. Deng et al., “Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles”. Proceedings of the National Academy of Sciences 113, 2868–2873 (2016). doi:10.1073/pnas.1520244113. ↩

  4. M. Sutter, D. Boehringer, S. Gutmann, S. Günther, D. Prangishvili, M.J. Loessner et al., “Structural basis of enzyme encapsulation into a bacterial nanocompartment”. Nature Structural & Molecular Biology 15, 939–947 (2008). doi:10.1038/nsmb.1473. ↩↩

  5. T.W. Giessen, B.J. Orlando, A.A. Verdegaal, M.G. Chambers, J. Gardener, D.C. Bell et al., “Large protein organelles form a new iron sequestration system with high storage capacity”. eLife 8, e46070 (2019). doi:10.7554/eLife.46070. ↩

  6. C. Cassidy-Amstutz, L. Oltrogge, C.C. Going, A. Lee, P. Teng, D. Quintanilla et al., “Identification of a minimal peptide tag for in vivo and in vitro loading of encapsulin”. Biochemistry 55, 3461–3468 (2016). doi:10.1021/acs.biochem.6b00294. ↩↩

  7. W.J. Altenburg, N. Rollins, P.A. Silver & T.W. Giessen, “Exploring targeting peptide-shell interactions in encapsulin nanocompartments”. Scientific Reports 11, 4951 (2021). doi:10.1038/s41598-021-84329-z. ↩↩

  8. P. Lagoutte et al., “Simultaneous surface display and cargo loading of encapsulin nanocompartments and their use for rational vaccine design”. Vaccine 36, 3622–3628 (2018). doi:10.1016/j.vaccine.2018.05.034. ↩

  9. J.A. Jones, A.S. Cristie-David, M.P. Andreas & T.W. Giessen, “Triggered reversible disassembly of an engineered protein nanocage”. Angewandte Chemie International Edition 60, 25034–25041 (2021). doi:10.1002/anie.202110318. ↩

  10. S. Kwon, M.P. Andreas, J.A. Jones & T.W. Giessen, “A permeable protein nanocage enables facile cargo loading and cytosolic protein delivery”. Nature Communications (2026), published 14 August 2026. doi:10.1038/s41467-026-76849-x. Earlier bioRxiv version. ↩↩

  11. S. Kwon, M.P. Andreas & T.W. Giessen, “Pore engineering as a general strategy to improve protein-based enzyme nanoreactor performance”. ACS Nano 18, 25740–25753 (2024). doi:10.1021/acsnano.4c08186. ↩

  12. S. Kwon & T.W. Giessen, “Engineered protein nanocages for concurrent RNA and protein packaging in vivo”. ACS Synthetic Biology 11, 3504–3515 (2022). doi:10.1021/acssynbio.2c00391. ↩↩↩↩

  13. R. Siddiquee, F. Lie, T.N. Szyszka, A. Loustau, M.P. Andreas, T.W. Giessen & Y.H. Lau, “Directed evolution of multimeric proteins is enabled by dual-compensatory gene duplication”. bioRxiv (2026), preprint version consulted. doi:10.64898/2026.01.12.698938. ↩↩

  14. N. Terasaka, Y. Azuma & D. Hilvert, “Laboratory evolution of virus-like nucleocapsids from nonviral protein cages”. Proceedings of the National Academy of Sciences 115, 5432–5437 (2018). doi:10.1073/pnas.1800527115. ↩

  15. S. Tetter, N. Terasaka, A. Steinauer, R.J. Bingham, S. Clark, A.J.P. Scott et al., “Evolution of a virus-like architecture and packaging mechanism in a repurposed bacterial protein”. Science 372, 1220–1224 (2021). doi:10.1126/science.abg2822. ↩

  16. P. Legault, J. Li, J. Mogridge, L.E. Kay & J. Greenblatt, “NMR structure of the bacteriophage λ N peptide/boxB RNA complex: recognition of a GNRA fold by an arginine-rich motif”. Cell 93, 289–299 (1998). doi:10.1016/S0092-8674(00)81579-2. ↩

  17. L.S. Qi, M.H. Larson, L.A. Gilbert, J.A. Doudna, J.S. Weissman, A.P. Arkin et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression”. Cell 152, 1173–1183 (2013). doi:10.1016/j.cell.2013.02.022. ↩

  18. K.M. Esvelt, J.C. Carlson & D.R. Liu, “A system for the continuous directed evolution of biomolecules”. Nature 472, 499–503 (2011). doi:10.1038/nature09929. ↩

  19. A. Ravikumar, G.A. Arzumanyan, M.K.A. Obadi, A.A. Javanpour & C.C. Liu, “Scalable, continuous evolution of genes at mutation rates above genomic error thresholds”. Cell 175, 1946–1957.e13 (2018). doi:10.1016/j.cell.2018.10.021. ↩

  20. C.S. Diercks, P. Sondermann, C. Rong, T.G. Gillis, Y. Ban, C. Wang et al., “An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli”. Science 389, 618–622 (2025). doi:10.1126/science.adp9583. ↩

  21. C.L. Moore, L.J. Papa & M.D. Shoulders, “A processive protein chimera introduces mutations across defined DNA regions in vivo”. Journal of the American Chemical Society 140, 11560–11564 (2018). doi:10.1021/jacs.8b04001. ↩