Efficient site-directed insertion of heterologous DNA into a genome remains an outstanding challenge. Recombinases that can integrate kilobase-sized DNA constructs are difficult to reprogram to user-defined loci, while genomic insertion using CRISPR-Cas methods relies on inefficient host DNA repair machinery. Here, we describe a Cas-Transposon (CasTn) system for genomic insertions that uses a Himar1 transposase fused to a catalytically dead dCas9 nuclease to mediate programmable, site-directed transposition. Using cell-free in vitro assays, we demonstrated that the Himar-dCas9 fusion protein increased the frequency of transposon insertion at a single targeted TA dinucleotide by >300-fold compared to a random transposase, and that site-directed transposition is dependent on target choice while robust to log-fold variations in protein and DNA concentrations. We also showed that Himar-dCas9 mediates directed transposition into plasmids in Escherichia coli. This work highlights CasTn as a new modality for host-independent, programmable, site-directed DNA insertions.

中文翻译：

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一种经过工程设计的Cas-Transposon系统，用于可编程和定点DNA转座。

有效地将异源DNA定点插入基因组仍然是一项艰巨的挑战。可以整合千碱基大小的DNA构建体的重组酶很难重编程为用户定义的基因座，而使用CRISPR-Cas方法进行基因组插入则依赖于效率低下的宿主DNA修复机制。在这里，我们描述了一个用于基因组插入的Cas-Transposon（CasTn）系统，该系统使用与催化死dCas9核酸酶融合的Himar1转座酶来介导可编程的定点转座。使用无细胞体外测定法，我们证明，与随机转座酶相比，Himar-dCas9融合蛋白使单个靶向TA二核苷酸处转座子插入的频率增加了300倍以上，而且这种定点转座取决于靶标的选择，同时对蛋白质和DNA浓度的对数变化也很稳定。我们还显示，Himar-dCas9介导定向转座入大肠杆菌中的质粒。这项工作强调了CasTn是一种独立于宿主的，可编程的，定点的DNA插入的新形式。