# Single-colony resolution of CRISPR–Cas adaptation in E. coli reveals altered spacer-source bias during solid-phase growth

## TL;DR

A new assay reveals how CRISPR–Cas adaptation in E. coli changes when bacteria grow on solid surfaces versus in liquid.

## Contribution

A semi-quantitative papillation assay enables single-colony detection of CRISPR adaptation in E. coli with high sensitivity.

## Key findings

- In solid-phase growth, only ~9% of spacers were plasmid-derived, compared to 64% in liquid culture.
- Adjusting inducer concentration did not restore plasmid bias, suggesting a physiological basis for the shift.
- The solid-phase adaptation reflects near-neutral DNA source sampling rather than plasmid overrepresentation.

## Abstract

CRISPR–Cas systems provide adaptive immunity by integrating short DNA fragments from mobile genetic elements into host arrays. While the core biochemical mechanism of adaptation is well defined, its modulation by physiological contexts is less well understood. Here, we present a visual papillation assay that enables single-colony detection of CRISPR–Cas adaptation in Escherichia coli. Spacer acquisition restores the reading frame of a disrupted lacZ gene, forming blue papillae on lactose X-gal plates. The assay is semi-quantitative, highly sensitive, capable of detecting single events among 109 cells, and responds predictably to Cas1–Cas2 expression levels. Spacer mapping revealed a major shift in source bias: in liquid culture, 64% of spacers were plasmid-derived, but on solid medium this dropped to ∼9%. Adjusting inducer concentration to match liquid conditions did not restore plasmid bias, indicating a physiological basis linked to colony growth. Accounting for the molar excess of chromosomal DNA, the 9% plasmid share reflects near-neutral DNA source sampling rather than plasmid overrepresentation. These findings suggest that the spatial and metabolic structure of colonies strongly shapes the adaptation landscape. The assay provides a scalable platform for dissecting condition-specific features of CRISPR–Cas adaptation, including spacer origin, sequence features, and growth context.

Graphical Abstract