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

Ido Yosef, Tridib Mahata, Moran G. Goren, Or J. Degany, Adam Ben-Shem, Udi Qimron*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)7552-7562
Number of pages11
JournalNucleic Acids Research
Volume51
Issue number14
DOIs
StatePublished - 11 Aug 2023

Funding

FundersFunder number
Horizon 2020 Framework Programme
European Research Council
Ministry of Health, State of Israel15370
Ministry of Health, State of Israel
Horizon 2020818878
Horizon 2020

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