Walker Matt W G, Klompe Sanne E, Zhang Dennis J, Sternberg Samuel H
Department of Biological Sciences, Columbia University, New York, NY, 10027, USA.
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, 10032, USA.
bioRxiv. 2023 Jan 19:2023.01.19.524723. doi: 10.1101/2023.01.19.524723.
CRISPR-associated transposons (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F CAST system. On the donor DNA, large mutagenic libraries identified core binding sites recognized by the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications of CAST systems.
CRISPR相关转座子（CASTs）利用核酸酶缺陷型CRISPR-Cas系统的RNA引导DNA结合活性，将DNA整合到靶位点下游。转座依赖于几种关键的蛋白质-蛋白质和蛋白质-DNA相互作用，但对于有效转座子DNA整合活性的明确序列要求知之甚少。在这里，我们利用汇集文库筛选和高通量测序来揭示I-F型CAST系统转座过程中的新序列决定因素。在供体DNA上，大型诱变文库鉴定出了TnsB转座酶识别的核心结合位点，以及一个额外的保守区域，该区域编码了整合宿主因子（IHF）的共有结合位点。值得注意的是，我们发现VchCAST需要IHF才能有效转座，从而揭示了一种参与CRISPR相关转座体组装的新型细胞因子。在靶DNA上，我们在整合位点发现了优先序列基序，以单碱基对分辨率解释了先前观察到的异质性。最后，我们利用文库数据设计了能够进行框内蛋白质标签的改良转座子变体。总的来说，我们的结果为TnsB与转座子DNA之间形成的双端复合物的组装和结构提供了新线索，并为CAST系统基因组工程应用中定制payload序列的设计提供了参考。
