Real-time Observation of CRISPR spacer acquisition by Cas1–Cas2 integrase
| Autor: | Fran Ding, Jagat B. Budhathoki, Yibei Xiao, Gabriel Schuler, Chunyi Hu, Alexander H.-D. Cheng, Ailong Ke |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Transcription
Genetic Computer science Information storage Cell lineage Computational biology Article chemistry.chemical_compound Structural Biology Transcription (biology) Enterococcus faecalis Escherichia coli Fluorescence Resonance Energy Transfer CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Molecular Biology biology Integrases Integrase Kinetics Förster resonance energy transfer Electroporation chemistry Mutation biology.protein Nucleic acid Microorganisms Genetically-Modified DNA |
| Zdroj: | Nature structural & molecular biology |
| ISSN: | 1545-9985 1545-9993 |
| Popis: | Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis (Efa) Cas1–Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1–Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes EfaCas1–Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex has a surprisingly short mean lifetime. Remarkably, transcription efficiently resolves the postsynaptic complex and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies. |
| Databáze: | OpenAIRE |
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