Understanding and engineering the microbiome.

In the 2019 study, engineered donor E. coli transferred DNA to resident gut bacteria in mice through direct cell-to-cell contact, a process called conjugation.

Depending on the delivery vector, the DNA could remain in the recipient as a plasmid, a DNA molecule separate from the chromosome, or be inserted into its chromosome. This allowed bacteria to be modified without first isolating and growing each recipient on its own.

Guide RNAs direct a CRISPR-associated protein complex to selected sites in bacterial DNA. Transposase proteins then insert a DNA payload nearby. The improved system described in the 2021 paper streamlined delivery and enabled insertions at multiple sites.

These insertions can be marker-free: a selectable marker does not need to be included in the inserted DNA, although transposon-associated sequences remain. The method does not require homologous recombination or a Cas9-induced double-strand break.

Engineered donor bacteria transfer the editing system through conjugation. Computationally designed guide RNAs direct CRISPR-associated transposases to insert DNA payloads at selected sites in particular bacteria within a community.

In mice, researchers added a metabolic pathway to a native gut bacterium and introduced reporter genes into segmented filamentous bacteria. These experiments demonstrated genetic access to gut bacteria that are difficult to grow in the laboratory.