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.

中文翻译：

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大肠杆菌Cas1 / 2核酸内切酶复合物降低CRISPR / Cascade导向阵列的稳定性

基于CRISPR的干扰在从遗传电路到动态代谢控制的各种应用中已经很普遍。在大肠杆菌中，可以通过删除cas3核酸酶以及引导RNA阵列的表达来将天然CRISPR Cascade系统用于沉默，其中可以从单个转录物中沉默多个基因。我们注意到用于动态沉默的引导阵列丢失了原间隔序列。我们报告不稳定的引导数组是由于Cas1 / 2核酸内切酶复合物的表达。cas1删除可改善引导阵列的稳定性。我们提出了一种模型，其中基础Cas1 / 2核酸内切酶活性导致引导阵列失去原间隔子。随后，可以通过选择扩增突变体引导阵列。