PT  - JOURNAL ARTICLE
AU  - Ye, Zhixia
AU  - Lebeau, Juliana
AU  - Moreb, Eirik A.
AU  - Menacho-Melgar, Romel
AU  - Lynch, Michael D.
TI  - The <em>E. coli</em> Cas1/2 endonuclease complex reduces CRISPR/Cascade guide array stability
AID  - 10.1101/2020.07.27.223214
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.07.27.223214
4099  - http://biorxiv.org/content/early/2020/07/27/2020.07.27.223214.short
4100  - http://biorxiv.org/content/early/2020/07/27/2020.07.27.223214.full
AB  - CRISPR 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 protospacer sequences from guide arrays utilized for dynamic silencing. We report that unstable guide arrays are due to expression of the Cas1/2 endonuclease complex. A cas1 deletion improves guide array stability. We propose a model wherein basal Cas1/2 endonuclease activity results in the loss of protospacers 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.HighlightsCas1/2 endonuclease complex mediates CRISPR/Cascade protospacer loss in E. coliTightly controlled Cascade operon expression increases guide array stability.Competing Interest StatementM.D. Lynch and Z. Ye have a financial interest in DMC Biotechnologies, Inc. M.D. Lynch, R Menacho-Melgar and E.A. Moreb have a financial interest in Roke Biotechnologies, LLC.