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Abstract
The CRISPR-Cas system functions as a prokaryotic immune system and is highly diverse, with six major types and numerous sub-types. The most abundant are type I CRISPR systems, which utilize a multi-subunit effector, Cascade, and a CRISPR RNA (crRNA) to detect invading DNA species. Detection leads to DNA loading of the Cas3 helicase-nuclease, leading to long-range deletions in the targeted DNA, thus providing immunity against mobile genetic elements (MGE). Here, we focus on the type I-G system, a streamlined, 4-subunit complex with an atypical Cas3 enzyme. We demonstrate that Cas3 helicase activity is not essential for immunity against MGE in vivo and explore applications of the Thioalkalivibrio sulfidiphilus Cascade effector for genome engineering in Escherichia coli. Long-range, bidirectional deletions were observed when the lacZ gene was targeted. Deactivation of the Cas3 helicase activity dramatically altered the types of deletions observed, with small deletions flanked by direct repeats that are suggestive of microhomology mediated end joining. When donor DNA templates were present, both the wild-type and helicase-deficient systems promoted homology-directed repair (HDR), with the latter system providing improvements in editing efficiency, suggesting that a single nick in the target site may promote HDR in E. coli using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.
Original language | English |
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Article number | 001373 |
Number of pages | 9 |
Journal | Microbiology |
Volume | 169 |
Issue number | 8 |
Early online date | 1 Aug 2023 |
DOIs | |
Publication status | Published - 1 Aug 2023 |
Keywords
- Cas3 helicase
- CRISPR
- Genome engineering
- Type I-G Cascade
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- 1 Finished
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Cyclic oligoadenylate signalling: Cyclic oligoadenylate signalling - a new type of antiviral response
White, M. (PI)
1/01/19 → 31/12/21
Project: Standard