@article{LiaoTtofaliSlotkowskietal.2019,
  author    = {Liao, Chunyu and Ttofali, Fani and Slotkowski, Rebecca A. and Denny, Steven R. and Cecil, Taylor D. and Leenay, Ryan T. and Keung, Albert J. and Beisel, Chase L.},
  title     = {Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis},
  series = {Nature Communications},
  volume    = {10},
  journal   = {Nature Communications},
  doi       = {10.1038/s41467-019-10747-3},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236843},
  year      = {2019},
  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.},
  language  = {en}
}