Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration.
Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration.
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转座子诱变文库揭示了 CRISPR RNA 引导的 DNA 整合过程中新的分子需求。
DOI：
10.1101/2023.01.19.524723
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Sternberg,SamuelH
中科院分区：
文献类型：
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作者：
Walker,MattWG;Klompe,SanneE;Zhang,DennisJ;Sternberg,SamuelH
crispr相关转座子（cast）利用rna引导的核酸酶缺陷CRISPR-Cas系统的DNA结合活性，直接靶位点下游的DNA整合。转座子依赖于几个关键的蛋白质-蛋白质和蛋白质-DNA相互作用，但对有效转座子DNA整合活性的显式序列要求知之甚少。在这里，我们利用汇集的文库筛选和高通量测序来揭示I-F型霍乱弧菌CAST系统转位过程中的新序列决定因素。在供体DNA上，大型诱变文库确定了TnsB转座酶识别的核心结合位点，以及编码整合宿主因子（IHF）一致结合位点的额外保守区域。值得注意的是，我们发现VchCAST需要IHF才能进行有效的转座，从而揭示了参与crispr相关转座体组装的一种新的细胞因子。在目标DNA上，我们发现了整合位点的首选序列基序，这解释了先前观察到的单碱基对分辨率的异质性。最后，我们利用我们的文库数据来设计修饰的转座子变体，使框内蛋白质标记成为可能。总的来说，我们的研究结果为TnsB和转座子DNA之间形成的成对末端复合物的组装和结构提供了新的线索，并为CAST系统基因组工程应用的定制有效载荷序列的设计提供了信息。
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 Vibrio cholerae 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.