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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 and explore applications of the Cascade effector for genome engineering in . Long-range, bidirectional deletions were observed when the 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 using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.

Funding
This study was supported by the:
  • China Scholarship Council (Award 202008060345)
    • Principle Award Recipient: QilinShangguan
  • Biotechnology and Biological Sciences Research Council (Award BB/S000313/1)
    • Principle Award Recipient: MalcolmF White
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2023-08-01
2024-12-02
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