Constructing efficient cellular factories often requires integration of heterologous pathways for synthesis of novel compounds and improved cellular productivity. Few genomic sites are routinely used, however, for efficient integration and expression of heterologous genes, especially in nonmodel hosts. Here, a data-guided framework for informing suitable integration sites for heterologous genes based on ATAC-seq was developed in the nonmodel yeast Komagataella phaffii. Single-copy GFP constructs were integrated using CRISPR/Cas9 into 38 intergenic regions (IGRs) to evaluate the effects of IGR size, intensity of ATAC-seq peaks, and orientation and expression of adjacent genes. Only the intensity of accessibility peaks was observed to have a significant effect, with higher expression observed from IGRs with low- to moderate-intensity peaks than from high-intensity peaks. This effect diminished for tandem, multicopy integrations, suggesting that the additional copies of exogenous sequence buffered the transcriptional unit of the transgene against effects from endogenous sequence context. The approach developed from these results should provide a basis for nominating suitable IGRs in other eukaryotic hosts from an annotated genome and ATAC-seq data.

中文翻译：

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使用 ATAC-seq 识别异源基因整合的改进位点。

构建高效的细胞工厂通常需要整合异源途径以合成新化合物并提高细胞生产力。然而，很少有基因组位点被常规用于异源基因的有效整合和表达，尤其是在非模型宿主中。在这里，在非模型酵母Komagataella phaffii中开发了一个基于 ATAC-seq 为异源基因提供合适整合位点的数据引导框架. 使用 CRISPR/Cas9 将单拷贝 GFP 构建体整合到 38 个基因间区域 (IGR) 中，以评估 IGR 大小、ATAC-seq 峰强度以及相邻基因的方向和表达的影响。仅观察到可达峰的强度具有显着影响，从具有低到中等强度峰的 IGR 中观察到的表达高于从高强度峰中观察到的表达。对于串联、多拷贝整合，这种效应减弱，这表明外源序列的额外拷贝缓冲了转基因的转录单位，以抵抗来自内源序列背景的影响。从这些结果开发的方法应该为从注释的基因组和 ATAC-seq 数据中提名其他真核宿主中合适的 IGR 提供基础。