Highly Active CRISPR-Adaptation Proteins Revealed by a Robust Enrichment Technology

Natural prokaryotic defense via the CRISPR-Cas system requires spacer integration into the CRISPR array in a process called adaptation. To examine if adaptation is regulated at the protein level, we established a robust Perpetual DNA Packaging and Transfer (PeDPaT) system that uses a strain of T7 phage to package plasmids and transfer them without killing the host, and then uses a different strain of T7 phage to repeat the cycle. We used PeDPaT to identify better adaptation proteins-Cas1 and Cas2-by enriching for mutants that provide better adaptation efficiency. We identified two mutant Cas1 proteins that show up to 10-fold enhanced adaptation. The mutants show higher integration and disintegration activity in vitro and one of them also binds DNA better<br> than the wild-type Cas1. Lastly, we showed that their specificity for selecting a protospacer adjacent motif (PAM) is decreased. The PeDPaT technology may be used for many robust screens requiring efficient and effortless DNA transduction.