3rd International Conference on CRISPR Technologies
Modular, One-Pot Assembly of CRISPR Arrays Enables Library Generation, Multi-Nuclease Targeting and Reveals Factors Influencing CRISPR RNA Biogenesis
Authors
Chunyu Liao - Presenter, Helmholtz Institute for RNA-based Infection Research
Fani Ttofali, North Carolina State University
Rebecca A. Slotkowski, North Carolina State University
Steven R. Denny, Columbia University
Ryan T. Leenay, North Carolina State University
Albert Keung, North Carolina State University
Chase Beisel, Helmholtz Institute for RNA-based Infection Research
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 âcompositeâ 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.
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 âcompositeâ 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.