Trials for in vivo DNA amplification of targeted region by dCas9-sgRNA-guided positioning of Primpol and Phi29 DNA polymerase

We attempted to generate linear extrachromosomal DNA to analyze long-distance transfer of linear DNA fragment. Our strategy is to amplify genomic region by positioning human primase/polymerase (Primpol) protein and/or bacteriophage phi29 DNA polymerase using deactivated Cas9 (dCas9)-single guide RNA (sgRNA). Here, we performed DNA-Seq of three lines of budding yeast; i) yeast harboring dCas9, sgRNA targeting yeast TEF1 promoter region, Primpol and phi29 DNA polymerase, ii) yeast harboring dCas9, sgRNA targeting yeast TEF1 promoter region and Primpol, and iii) yeast harboring none of these four components.