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

Author

Listed:
  • Chunyu Liao

    (North Carolina State University
    Helmholtz Institute for RNA-based Infection Research)

  • Fani Ttofali

    (North Carolina State University)

  • Rebecca A. Slotkowski

    (North Carolina State University)

  • Steven R. Denny

    (North Carolina State University)

  • Taylor D. Cecil

    (North Carolina State University)

  • Ryan T. Leenay

    (North Carolina State University)

  • Albert J. Keung

    (North Carolina State University)

  • Chase L. Beisel

    (North Carolina State University
    Helmholtz Institute for RNA-based Infection Research
    University of Würzburg)

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.

Suggested Citation

  • Chunyu Liao & Fani Ttofali & Rebecca A. Slotkowski & Steven R. Denny & Taylor D. Cecil & Ryan T. Leenay & Albert J. Keung & Chase L. Beisel, 2019. "Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-10747-3
    DOI: 10.1038/s41467-019-10747-3
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    Cited by:

    1. Dixit, Yatika & Yadav, Preeti & Sharma, Arun Kumar & Pandey, Poornima & Kuila, Arindam, 2023. "Multiplex genome editing to construct cellulase engineered Saccharomyces cerevisiae for ethanol production from cellulosic biomass," Renewable and Sustainable Energy Reviews, Elsevier, vol. 187(C).

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