Highly active CRISPR-adaptation proteins revealed by a robust enrichment technology - Archive ouverte HAL
Article Dans Une Revue Nucleic Acids Research Année : 2023

Highly active CRISPR-adaptation proteins revealed by a robust enrichment technology

Résumé

Natural prokaryotic defense via the CRISPR–Cas system requires spacer integration into the CRISPR array in a process called adaptation. To search for adaptation proteins with enhanced capabilities, 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 mutants that provide higher adaptation efficiency. We identified two mutant Cas1 proteins that show up to 10-fold enhanced adaptation in vivo. In vitro, one mutant has higher integration and DNA binding activities, and another has a higher disintegration activity compared to the wild-type Cas1. Lastly, we showed that their specificity for selecting a protospacer adjacent motif is decreased. The PeDPaT technology may be used for many robust screens requiring efficient and effortless DNA transduction.

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Génétique
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hal-04752786 , version 1 (24-10-2024)

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Ido Yosef, Tridib Mahata, Moran G Goren, Or J Degany, Adam Ben Shem, et al.. Highly active CRISPR-adaptation proteins revealed by a robust enrichment technology. Nucleic Acids Research, 2023, 51 (14), pp.7552-7562. ⟨10.1093/nar/gkad510⟩. ⟨hal-04752786⟩
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