Cas1/2 Endonuclease Complex Modifies Self-Targeting CRISPR/Cascade Spacers Reducing Silencing Guide Stability.

CRISPR-based interference has become common in various applications from genetic circuits to dynamic metabolic control. In , the native CRISPR Cascade system can be utilized for silencing by deletion of the 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.