iDEC 2026 · Evolution Suisse
Encapsulins
Continuous directed evolution of a protein nanocompartment, with survival coupled to how well the cell packages dCas9 and its guide RNA.
- Target
- QtEncapsulin
6NJ8 - Platform
- MutaT7 continuous evolution
- Selection
- dCas9 sequestration → survival
Background
The evolution runs inside a single continuously dividing E. coli strain carrying two plasmids under MutaT7. What is being evolved is QtEncapsulin, the nanocompartment drawn below; what decides whether a cell keeps growing is whether its shell can catch and hold a dCas9·sgRNA complex before that complex silences an antibiotic-resistance gene. The three acts that follow walk through one cell, one cycle at a time.
How the selection works
01
Silence
dCas9 binds inside the resistance gene and throttles it.
A catalytically dead Cas9, guided into the kanamycin-resistance ORF, obstructs rather than cuts. Elongating RNA polymerase collides with the R-loop, is delayed, and eventually displaces it.
Repression is kinetic, not absolute: the locus is throttled rather than switched off, and resistance settles below what the kanamycin in the medium demands. The guide targets the gene body, not the promoter — that choice is what makes the output graded rather than binary.
02
Diversify
MutaT7 mutates one cassette and nothing else.
A cytidine deaminase fused to T7 RNA polymerase mutates processively from a T7 promoter to its terminator. The encapsulin ORF is the only sequence inside that window.
The host genome, replicated by its own polymerases, is untouched. Diversity accumulates in growing culture — no transformation rounds, no step performed in a tube.
03
Encapsulate
A shell that captures the repressor restores resistance.
240 subunits assemble into a T=4 compartment 42 nm across. dCas9 carries the encapsulin's native cargo-loading peptide; the sgRNA carries a boxB hairpin, caught by a λN peptide grafted onto the inner surface. Capturing either half clears the gene.
Growth rate is then a readout of encapsulation efficiency, and continuous culture integrates that margin over many generations. Raising the kanamycin concentration between passages raises the fraction that must be captured.
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