|
| Status |
Public on Feb 03, 2020 |
| Title |
Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas |
| Organism |
synthetic construct |
| Experiment type |
Other
|
| Summary |
Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids that are encountered, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that casposase integration in vitro recapitulates several properties of CRISPR-Cas integrases. The X-ray structure of Methanosarcina mazei casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization and the separation of Cas1 dimers. This, in turn, led to preferred integration of single spacers over two transposon ends.
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| |
|
| Overall design |
Next generation sequencing experiments following casposon integration assay in order to identify / characterize integrations site, motifs and any poteintial off-target sites
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| |
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| Contributor(s) |
Hickman AB, Kailasan S, Genzor P, Haase AD, Dyda F |
| Citation(s) |
31913120 |
| |
| Submission date |
Oct 17, 2019 |
| Last update date |
Feb 03, 2020 |
| Contact name |
Pavol Genzor |
| E-mail(s) |
pavol.genzor@gmail.com
|
| Organization name |
Henry M Jackson Foundation
|
| Department |
ACESO
|
| Street address |
6720A Rockledge Drive, Suite 100
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20817 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL17769 |
Illumina MiSeq (synthetic construct) |
|
| Samples (5)
|
|
| Relations |
| BioProject |
PRJNA578121 |
| SRA |
SRP226077 |
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