Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Nucleic Acids Res. 2023 May 22;51(9):4519-4535. doi: 10.1093/nar/gkad270.
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.
CRISPR 相关转座酶（CASTs）利用无核酸酶活性的 CRISPR-Cas 系统的 RNA 引导的 DNA 结合活性，在靶位点下游指导 DNA 整合。转座依赖于几个关键的蛋白-蛋白和蛋白-DNA 相互作用，但对于控制转座子 DNA 整合活性的明确序列要求知之甚少。在这里，我们利用池库筛选和高通量测序来揭示 I 型-F 霍乱弧菌 CAST 系统（VchCAST）转座过程中的新序列决定因素。在供体 DNA 上，大型转座子末端文库揭示了 TnsB 转座酶的结合位点核苷酸偏好性，以及编码整合宿主因子（IHF）整合位点保守序列的额外区域。值得注意的是，我们发现 VchCAST 为了高效转座需要 IHF，从而揭示了一个新的参与 CRISPR 相关转座体组装的细胞因子。在靶 DNA 上，我们在整合位点发现了偏好的序列基序，以单碱基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修饰的转座子变体，实现了蛋白质的框内标记。总的来说，我们的结果为 TnsB 和转座子 DNA 之间形成的末端复合物的组装和结构提供了新的线索，并为 CAST 系统的基因组工程应用设计定制有效载荷序列提供了信息。
