Massively Parallel Screens Reveal Factors Influencing the Activity of a Diversity-Generating Retroelement Reconstituted in Escherichia coli [TranscriptionalStrength]

Some phages and microbes employ Diversity-Generating Retroelements (DGRs) to achieve rapid targeted adaptation. DGRs mutagenize a specific gene without harming the rest of the genome. Mutagenesis relies on the DGR reverse transcriptase (dRT), which is highly error-prone when transcribing adenines of an RNA template. The resulting mutagenized cDNA then alters the target gene through a poorly understood mechanism. Transplanting DGRs into a tractable organism, like Escherichia coli, holds promise for creating a powerful system for gene-targeted editing. Here, we successfully reconstituted the archetypal BPP DGR in E. coli, demonstrating that DGRs are "plug-and-play" systems adaptable to non-native hosts. Systematic analysis of reconstituted DGR established principles for target mutagenesis. Our results revealed that cytosine misincorporation is the most common error when dRT transcribes adenines. However, error rates peak in 5'-AAC-3' and 5'-ACC-3' contexts, driven by elevated adenine and guanosine misincorporations, respectively. Using high-throughput genetics, we discovered that cells lacking the single-stranded DNA exonuclease ExoI exhibit 15-fold higher DGR activity. Our evidence indicates that ExoI inhibits DGRs by binding a limited cellular factor, rather than through direct cDNA degradation. Finally, by profiling DGR targets across thousands of chromosomal loci, we discovered that DGRs preferentially edit targets near the replication origin and oriented in the same direction as replication. We showed that the directionality of replication underlies this bias, possibly through unwinding the target to facilitate base-pairing with incoming cDNA. These findings establish a reconstituted DGR system in E. coli and provide a foundation for optimizing targeted gene-editing applications in the future. Overall design: DGR reporter integrated in the chromosome at 3758105 under the control of either a strong (J23118) or weak (J23112) promoter. Activated DGR repair to and sequence for mutagenesis rates to test whether the strength of the target's promoter affects repair frequency.

部分噬菌体与微生物利用多样性生成逆转录元件（Diversity-Generating Retroelements, DGRs）实现快速的定向适应性演化。DGRs可对特定基因进行诱变而不损伤基因组其余区域。该诱变过程依赖于DGR逆转录酶（DGR reverse transcriptase, dRT），该酶在转录RNA模板中的腺嘌呤时具有极高的错配倾向。所产生的经诱变的互补DNA（cDNA）随后通过一种尚未完全阐明的机制改变靶基因。将DGRs移植到如大肠杆菌（Escherichia coli）这类易操作的模式生物中，有望构建出强大的基因靶向编辑系统。本研究成功在大肠杆菌中重构了典型的BPP DGR系统，证实DGRs属于即插即用型系统，可适配非天然宿主。通过对重构后的DGR进行系统性分析，我们确立了靶基因诱变的相关原则。研究结果显示，当dRT转录腺嘌呤时，胞嘧啶的错掺入是最为常见的错误类型。不过，错配率在5'-AAC-3'与5'-ACC-3'序列背景下达到峰值，这分别由腺嘌呤与鸟嘌呤的错掺入水平升高所驱动。借助高通量遗传学手段，我们发现缺失单链DNA外切酶ExoI的细胞，其DGR活性提升了15倍。相关证据表明，ExoI通过结合有限的细胞因子抑制DGR活性，而非直接降解cDNA。最后，通过对数千个染色体位点的DGR靶标进行分析，我们发现DGR优先编辑复制起点附近且与复制方向同向的靶标。我们证实，复制的方向性是该偏好性的基础，其可能通过解开靶标双链以促进其与进入的互补DNA链进行碱基配对。本研究构建的大肠杆菌DGR重构系统，为未来优化靶向基因编辑应用奠定了基础。整体实验设计：将DGR报告基因整合于染色体3758105位点，受强启动子（J23118）或弱启动子（J23112）调控。通过检测活化的DGR修复效率与诱变序列速率，以验证靶基因启动子强度是否会影响修复频率。