Harnessing type I CRISPR–Cas systems for genome engineering in human cells
| Autor: | Peter Cameron, Mckenzi S. Toh, Steven B. Kanner, John van der Oost, Scott Gradia, Bastien Vidal, Carolyn Williams, Samuel H. Sternberg, Lynda M. Banh, Alexandra M. Lied, Paul Daniel Donohoue, David B. Nyer, Chris R. Fuller, B. Kohrs, Mary M. Coons, Matthew J. Irby, Chun Han Lin, Leslie S. Edwards, Arthur L.G. Owen, Tim Künne, Tomer Rotstein, Sanne E. Klompe, Rachel Kennedy, Euan M. Slorach, Andrew May, Stan J. J. Brouns, Stephen C. Smith |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Biomedical Engineering
Bioengineering Computational biology Applied Microbiology and Biotechnology Microbiology Genome engineering 03 medical and health sciences 0302 clinical medicine Plasmid Genome editing Microbiologie Escherichia coli Life Science Humans CRISPR Guide RNA 030304 developmental biology VLAG Gene Editing 0303 health sciences Nuclease Genome Models Genetic biology HEK 293 cells BacGen HEK293 Cells biology.protein Molecular Medicine Heterologous expression CRISPR-Cas Systems 030217 neurology & neurosurgery Biotechnology |
| Zdroj: | Nature Biotechnology Nature Biotechnology, 37, 1471-1477 Nature Biotechnology 37 (2019) |
| ISSN: | 1087-0156 |
| DOI: | 10.1038/s41587-019-0310-0 |
| Popis: | Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5–7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8–11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells. A type I CRISPR–Cas system, rather than the conventional type II CRISPR systems, is adapted for gene editing by fusing Cascade to the FokI nuclease domain. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink