Cells must detect and respond to molecular events such as the presence or absence of specific small molecules. To accomplish this, cells have evolved methods to measure the presence and concentration of these small molecules in their environment and enact changes in gene expression or behavior. However, cells don’t usually change their DNA in response to such outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into a genomic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Therefore, through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered plasmids, which correspond to two orthogonal small molecule inducers that can be recorded with this system. We identified the optimal location of guide RNA binding to the Bxb1 attP integrase attachment site, and characterized the detection limits of the system by measuring the minimal small molecule concentration and shortest induction time necessary to produce measurable differences in array composition as read out by Oxford Nanopore long read sequencing technology.

中文翻译：

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活细胞中连续事件记录的概念证明

细胞必须检测并响应分子事件，例如特定小分子的存在或不存在。为了实现这一点，细胞已经进化出方法来测量这些小分子在其环境中的存在和浓度，并改变基因表达或行为。然而，细胞通常不会响应这种外部刺激而改变它们的 DNA。在这项工作中，我们设计了一个基因回路，可以响应小分子浓度的变化而产生特定和受控的基因变化。已知的 DNA 序列可以重复整合到基因组阵列中，以便它们的身份和顺序编码有关细胞过去经历的小分子浓度的信息。为了实现这一点，我们使用无催化活性的 CRISPR-Cas9 (dCas9) 结合并阻断整合酶 Bxb1 的附着位点。因此，通过 dCas9 和向导 RNA 的共表达，Bxb1 可以被引导整合两个工程质粒之一，这对应于可以用该系统记录的两个正交小分子诱导剂。我们确定了引导 RNA 与 Bxb1 attP 整合酶附着位点结合的最佳位置，并通过测量最小小分子浓度和最短诱导时间来表征系统的检测限，以产生由牛津纳米孔读出的阵列组成的可测量差异长读长测序技术。这对应于可以用该系统记录的两个正交小分子诱导剂。我们确定了引导 RNA 与 Bxb1 attP 整合酶附着位点结合的最佳位置，并通过测量最小小分子浓度和最短诱导时间来表征系统的检测限，以产生由牛津纳米孔读出的阵列组成的可测量差异长读长测序技术。这对应于可以用该系统记录的两个正交小分子诱导剂。我们确定了引导 RNA 与 Bxb1 attP 整合酶附着位点结合的最佳位置，并通过测量最小小分子浓度和最短诱导时间来表征系统的检测限，以产生由牛津纳米孔读出的阵列组成的可测量差异长读长测序技术。