TY  - JOUR
A1  - Liao, Chunyu
A1  - Ttofali, Fani
A1  - Slotkowski, Rebecca A.
A1  - Denny, Steven R.
A1  - Cecil, Taylor D.
A1  - Leenay, Ryan T.
A1  - Keung, Albert J.
A1  - Beisel, Chase L.
T1  - Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis
JF  - Nature Communications
N2  - 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.
KW  - biotechnology
KW  - CRISPR-Cas systems
KW  - microbiology
KW  - small RNAs
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236843
VL  - 10
ER  -