Attributions#
Reused material#
Plasmids#
| Source | Obtained as | Used for |
|---|---|---|
pdCas9-bacteria (Addgene) |
Bacterial stab | Source of the dCas9 coding sequence, amplified out by PCR |
pSC101-T7-T3RNAP (Addgene) |
Bacterial stab | MutaT7 platform components |
| pDB series (Addgene) | Bacterial stab | Mutagenesis plasmid backbone |
pET-Duet-1 |
Laboratory stock | Expression backbone for shell constructs |
Published work#
The project builds on the following published work: - QtEncapsulin's structure and native cargo-loading chemistry Giessen et al., eLife 8, e46070 (2019). - Shell permeability and single-step cargo loading — Kwon, Andreas, Jones & Giessen, bioRxiv (2026), doi:10.64898/2026.04.06.716810. - Directed evolution of this shell, and the dominant-negative problem it raises — Siddiquee et al., bioRxiv (2026), doi:10.64898/2026.01.12.698938. - The λN·boxB handle and evolution of an RNA-packaging capsid — Tetter et al., Science 372, 1220–1224 (2021), doi:10.1126/science.abg2822. - Guide-truncation titration of dCas9 repression, and the kick-out model our selection depends on — Vigouroux et al., Molecular Systems Biology 14, e7899 (2018), doi:10.15252/msb.20177899. - MutaT7: a T7 RNA polymerase–deaminase fusion for targeted hypermutation — Moore, Papa & Shoulders, J. Am. Chem. Soc. 140, 11560 (2018). - Continuous hypermutation for context and comparison — Diercks et al., Science 389, 618–622 (2025), doi:10.1126/science.adp9583.
The project combines these components in a CRISPRi circuit that couples encapsulation to survival and gives one shell two orthogonal handles on a ribonucleoprotein.
Protocols#
Published protocols were used and modified where the equipment differed. The Ni-NTA procedure was adapted from a manufacturer protocol for pre-packed columns, a published methods section and local practice for hand-packed columns. Modifications are documented in Protocols.
Software and infrastructure#
Plasmid design and sequence alignment were done in Benchling. Synthetic gene fragments were obtained from Twist Bioscience, and primers and whole-plasmid sequencing from Microsynth. This wiki is built with MkDocs. Protein and RNA illustrations were rendered with UCSF ChimeraX from PDB 5F9R (Cas9–guide RNA) and PDB 1MSW (T7 polymerase and nascent RNA). The overview shows the Cas9 and guide separately; Mechanism retains the deposited complexes. Active Cas9 serves as a structural illustration of dCas9; the engineered CLP, boxB and deaminase fusions are not present in these deposited structures.
Acknowledgments#
- Daria Belous and Annabelle Winzer, supervisors, for guidance and expertise; Daria Belous also provided the MutaT7 plasmid.
- Prof. Dr. Kathrin Lang and the Lang group, for laboratory space, equipment and resources. Dr. Maximilian Fottner, Dr. Vera Wanka and Paul Schnacke answered questions throughout the project.
- Student BioLab Zürich, for hosting the team.
- Kian Bigović Villi and Philip Nitsch, advisors throughout.
- The Hilvert Lab, for guidance during the initial development of the idea.
- Dr. Mikail Levasseur, for assistance and insight during cage purification.
- Microsynth AG, Universität Zürich and ETH Zürich, for financial support; Microsynth AG also provided sequencing and oligonucleotides (see Team and sponsors).