Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis.
dc.contributor.author | Liao, Chunyu | |
dc.contributor.author | Ttofali, Fani | |
dc.contributor.author | Slotkowski, Rebecca A | |
dc.contributor.author | Denny, Steven R | |
dc.contributor.author | Cecil, Taylor D | |
dc.contributor.author | Leenay, Ryan T | |
dc.contributor.author | Keung, Albert J | |
dc.contributor.author | Beisel, Chase L | |
dc.date.accessioned | 2019-08-19T13:12:35Z | |
dc.date.available | 2019-08-19T13:12:35Z | |
dc.date.issued | 2019-07-03 | |
dc.identifier.citation | Nat Commun. 2019 Jul 3;10(1):2948. doi: 10.1038/s41467-019-10747-3. | en_US |
dc.identifier.issn | 2041-1723 | |
dc.identifier.pmid | 31270316 | |
dc.identifier.doi | 10.1038/s41467-019-10747-3 | |
dc.identifier.uri | http://hdl.handle.net/10033/621909 | |
dc.description.abstract | CRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis. | en_US |
dc.language.iso | en | en_US |
dc.publisher | Springer-Nature | en_US |
dc.rights | Attribution-NonCommercial-ShareAlike 4.0 International | * |
dc.rights.uri | http://creativecommons.org/licenses/by-nc-sa/4.0/ | * |
dc.title | Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis. | en_US |
dc.type | Article | en_US |
dc.contributor.department | HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. | en_US |
dc.identifier.journal | Nature Communications | en_US |
refterms.dateFOA | 2019-08-19T13:12:36Z | |
dc.source.journaltitle | Nature communications |
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publications of the research group biology of synthetic RNA ([HIRI] RSYN) [22]
Examination and utilization of CRISPER-CAS systems