Journal
NUCLEIC ACIDS RESEARCH
Volume 49, Issue 11, Pages 6347-6363Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkab348
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Funding
- New Energy and Industrial Technology Development Organization (NEDO)
- Program on Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA)
- Japan Science and Technology Agency
- Japan Science and Technology Agency (JST), Adaptable and Seamless Technology transfer Program through Target-driven RD (A-STEP)
- Japan Science and Technology Agency, Adaptable and Seamless Technology transfer Program through Target-driven RD
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The study identified Cas10d as a functional nuclease in the CRISPR-Cas type I-D system, enabling targeted mutagenesis of genomic DNA in human cells. This suggests the potential repurposing of the CRISPR-Cas type I-D system as a unique effector pathway for genome engineering in eukaryotic cells.
Adoption of CRISPR-Cas systems, such as CRISPR-Cas9 and CRISPR-Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I-the most abundant CRISPR system in bacteria-remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense ('Cascade': Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR-Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR-Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR-Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.
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