Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis

Chunyu Liao, Fani Ttofali, Rebecca A. Slotkowski, Steven R. Denny, Taylor D. Cecil, Ryan T. Leenay, Albert J. Keung and Chase L. Beisel ()
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Chunyu Liao: North Carolina State University
Fani Ttofali: North Carolina State University
Rebecca A. Slotkowski: North Carolina State University
Steven R. Denny: North Carolina State University
Taylor D. Cecil: North Carolina State University
Ryan T. Leenay: North Carolina State University
Albert J. Keung: North Carolina State University
Chase L. Beisel: North Carolina State University

Nature Communications, 2019, vol. 10, issue 1, 1-14

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-10747-3

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