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

• DOI: 10.1038/s41467-019-10747-3
• Publication Date: 2019-07-03T10:03:04Z
• Authors: Chunyu Liao; Fani Ttofali; Rebecca A. Slotko...; Steven R. Denny; Taylor D. Cecil; Ryan T. Leenay; Albert J. Keung; Chase L. Beisel
• Keywords: Trans-activating crRNA; Guide RNA; Multiplex; CRISPR interference

Abstract CRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, sensing. However, arrays remain difficult generate due their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES construct and array libraries. allows assembly of repeat-spacer subunits using defined junctions within the trimmed portion spacers. Using CRATES, for single-effector nucleases Cas9, Cas12a, Cas13a that mediated multiplexed DNA/RNA cleavage gene regulation in cell-free systems, bacteria, yeast. further construction libraries composite utilized by multiple Cas nucleases. Finally, characterization reveals processing extraneous RNAs from Cas12a terminal repeats sequence- context-dependent loss RNA-directed nuclease activity via global RNA structure formation. thus can facilitate diverse multiplexing applications help identify factors impacting crRNA biogenesis.