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Genome editing in mammalian cells using the CRISPR type I-D nuclease.

Nucleic acids research | 2021

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.

Pubmed ID: 34076237 RIS Download

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BLASTN (tool)

RRID:SCR_001598

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Promega (tool)

RRID:SCR_006724

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RRID:SCR_008395

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Thermo Fisher Scientific (tool)

RRID:SCR_008452

Commercial vendor and service provider of laboratory reagents and antibodies. Supplier of scientific instrumentation, reagents and consumables, and software services.

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QIAGEN (tool)

RRID:SCR_008539

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Millipore (tool)

RRID:SCR_008983

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Mascot (tool)

RRID:SCR_014322

A software package and server used to identify and characterize proteins from primary sequence databases using mass spectrometry data. Mascot integrates peptide mass fingerprinting, sequence querying, and MS/MS ion searching in order to search for proteins in databases like SwissProt, NCBInr, EMBL EST divisions, contaminants, and cRAP. If a license is purchased, users may: search data sets that exceed the 1200 spectrum limit of the free version; set up automated, high throughput work; add and edit proteins and quantification methods; and search a preferred collection of sequence databases. The software package works with instruments from AB Sciex, Agilent, Bruker, Jeol, Shimadzu, Thermo Scientific, and Waters.

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RRID:SCR_014345

Software for MS/MS proteomic experiments to compare samples, identify biological relevance, and identify isoforms and protein PTMs. These proteins can be classified based on molecular function or organelle. Users can investigate spectrum details and counts, as well as use high through-put batch processing. Tutorials and a free trial are available through the main site.

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RRID:SCR_014565

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RRID:SCR_017055

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RRID:SCR_017277

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RRID:CVCL_0063

Cell line HEK293T is a Transformed cell line with a species of origin Homo sapiens (Human)

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HEK293T (tool)

RRID:CVCL_0063

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