CRISPR-based technologies have become central to genome engineering. However, CRISPR-based editing strategies are dependent on the repair of DNA breaks via endogenous DNA repair mechanisms, which increases susceptibility to unwanted mutations. Here we complement Cas9 with a recombinase's functionality by fusing a hyperactive mutant resolvase from transposon Tn3, a member of serine recombinases, to a catalytically inactive Cas9, which we term integrase Cas9 (iCas9). We demonstrate iCas9 targets DNA deletion and integration. First, we validate iCas9's function in Saccharomyces cerevisiae using a genome-integrated reporter. Cooperative targeting by CRISPR RNAs at spacings of 22 or 40 bp enables iCas9-mediated recombination. Next, iCas9's ability to target DNA deletion and integration in human HEK293 cells is demonstrated using dual GFP-mCherry fluorescent reporter plasmid systems. Finally, we show that iCas9 is capable of targeting integration into a genomic reporter locus. We envision targeting and design concepts of iCas9 will contribute to genome engineering and synthetic biology.

中文翻译：

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RNA引导的重组酶-Cas9融合靶向基因组DNA的缺失和整合。

基于CRISPR的技术已成为基因组工程的核心。然而，基于CRISPR的编辑策略取决于通过内源性DNA修复机制对DNA断裂的修复，从而增加了对有害突变的敏感性。在这里，我们通过将转座子Tn3（一种丝氨酸重组酶的成员）的高活性突变型分解酶融合到催化失活的Cas9（我们称之为整合Cas9（iCas9））上，使Cas9具有重组酶的功能。我们证明iCas9目标DNA缺失和整合。首先，我们使用基因组整合的报告子验证iCas9在酿酒酵母中的功能。CRISPR RNA以22或40 bp的间隔进行合作靶向使iCas9介导的重组成为可能。接下来，iCas9' 使用双重GFP-mCherry荧光报告质粒系统证明了在人HEK293细胞中靶向DNA缺失和整合的能力。最后，我们证明iCas9能够靶向整合到基因组报告基因座中。我们设想iCas9的靶向和设计概念将有助于基因组工程和合成生物学。