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

Chunyu LiaoFani TtofaliRebecca A SlotkowskiSteven R DennyTaylor D CecilRyan T LeenayAlbert J KeungChase L Beisel
Published in: Nature communications (2019)
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.
Keyphrases
  • genome editing
  • crispr cas
  • high density
  • cell free
  • high resolution
  • high throughput
  • circulating tumor
  • gene expression
  • dna binding
  • dendritic cells
  • regulatory t cells
  • genome wide
  • amino acid