Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis
Please always quote using this URN: urn:nbn:de:bvb:20-opus-236843
- 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-effectorCRISPR-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.…
Author: | Chunyu Liao, Fani Ttofali, Rebecca A. Slotkowski, Steven R. Denny, Taylor D. Cecil, Ryan T. Leenay, Albert J. Keung, Chase L. Beisel |
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URN: | urn:nbn:de:bvb:20-opus-236843 |
Document Type: | Journal article |
Faculties: | Medizinische Fakultät / Institut für Molekulare Infektionsbiologie |
Language: | English |
Parent Title (English): | Nature Communications |
Year of Completion: | 2019 |
Volume: | 10 |
Article Number: | 2948 |
Source: | Nature Communications (2019) 10:2948. https://doi.org/10.1038/s41467-019-10747-3 |
DOI: | https://doi.org/10.1038/s41467-019-10747-3 |
Dewey Decimal Classification: | 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit |
Tag: | CRISPR-Cas systems; biotechnology; microbiology; small RNAs |
Release Date: | 2024/07/11 |
Licence (German): | CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International |