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Download fileEscherichia coli Cas1/2 Endonuclease Complex Modifies Self-Targeting CRISPR/Cascade Spacers Reducing Silencing Guide Stability
journal contribution
posted on 17.12.2020, 13:38 authored by Zhixia Ye, Eirik A. Moreb, Shuai Li, Juliana Lebeau, Romel Menacho-Melgar, Matthew Munson, Michael D. LynchCRISPR-based interference has become
common in various applications
from genetic circuits to dynamic metabolic control. In E. coli, the native CRISPR Cascade system can be
utilized for silencing by deletion of the cas3 nuclease
along with expression of guide RNA arrays, where multiple genes can
be silenced from a single transcript. We notice the loss of spacer
sequences from guide arrays utilized for dynamic silencing. We report
that unstable guide arrays are due to expression of the Cas1/2 endonuclease
complex. We propose a model wherein basal Cas1/2 endonuclease activity
results in the loss of spacers from guide arrays. Subsequently, mutant
guide arrays can be amplified through selection. Replacing a constitutive
promoter driving Cascade complex expression with a tightly controlled
inducible promoter improves guide array stability, while minimizing
leaky gene silencing. Additionally, these results demonstrate the
potential of Cas1/2 mediated guide deletion as a mechanism to avoid
CRISPR based autoimmunity.