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Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis.
Liao, Chunyu Ttofali, Fani Slotkowski, Rebecca A Denny, Steven R Cecil, Taylor D Leenay, Ryan T Keung, Albert J Beisel, Chase L
Liao, Chunyu
Ttofali, Fani
Slotkowski, Rebecca A
Denny, Steven R
Cecil, Taylor D
Leenay, Ryan T
Keung, Albert J
Beisel, Chase L
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2019-07-03
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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.
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Nat Commun. 2019 Jul 3;10(1):2948. doi: 10.1038/s41467-019-10747-3.
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en
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2041-1723
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Attribution-NonCommercial-ShareAlike 4.0 International