Modular, One-Pot Assembly of CRISPR Arrays Enables Library Generation, Multi-Nuclease Targeting and Reveals Factors Influencing CRISPR RNA Biogenesis

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 constructed 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 allowed the one-pot
construction of array libraries and “composite” arrays utilized by multiple Cas nucleases. Finally,
array characterization revealed processing of extraneous CRISPR RNAs from Cas12a terminal
repeats and sequence- and context-dependent loss of crRNA-directed nuclease activity via global
RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help
identify factors impacting crRNA biogenesis.