Novel molecular requirements for CRISPR RNA-guided transposition.
Novel molecular requirements for CRISPR RNA-guided transposition.
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DOI：
10.1093/nar/gkad270
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发表时间：
2023-05-22
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
--
作者：
CRISPR相关转座酶（CAST）使用核酸酶缺陷型CRISPR-Cas系统的RNA引导的DNA结合活性指导靶位点下游的DNA整合。转座依赖于几个关键的蛋白质-蛋白质和蛋白质-DNA相互作用，但很少有人知道的明确的序列要求，有效的转座子DNA整合活动。在这里，我们利用汇集库筛选和高通量测序，以揭示新的序列决定因素在转座过程中的I-F型霍乱弧菌CAST系统（VchCAST）。在供体DNA上，大的转座子末端文库揭示了TnsB转座酶的结合位点核苷酸偏好，以及编码整合宿主因子（IHF）的共有结合位点的另外的保守区域。值得注意的是，我们发现VchCAST需要IHF进行有效转座，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选的序列基序，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，使框内蛋白质标签。总的来说，我们的研究结果提供了关于TnsB和转座子DNA之间形成的配对末端复合物的组装和结构的新线索，并为CAST系统的基因组工程应用提供了定制有效载荷序列的设计。汇集的DNA文库揭示了TnsB转座酶的核苷酸偏好，允许设计用于框内蛋白标记的功能性接头序列，并揭示了整合宿主因子（IHF）的参与。
CRISPR-associated transposases (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 (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for 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 with CAST systems. Pooled DNA libraries reveal nucleotide preferences for the TnsB transposase, permit the design of functional linker sequences for in-frame protein tagging, and uncover the involvement of integration host factor (IHF).
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改进的荧光蛋白用于内源性蛋白质标记。
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