In vivo diversification of target genomic sites using processive T7 RNA polymerase-base deaminase fusions blocked by RNA-guided dCas9

Abstract

Diversification of specific DNA segments typically involve in vitro generation of large sequence libraries and their introduction in cells for selection. Alternative in vivo mutagenesis systems on cells often show deleterious offsite mutations and restricted capabilities. To overcome these limitations, we have developed an in vivo platform to diversify specific DNA segments based on protein fusions between various base deaminases (BD) and the T7 RNA polymerase (T7RNAP) that recognizes a cognate promoter oriented towards the target sequence. The transcriptional elongation of these fusions generates transitions C to T or A to G on both DNA strands and in long DNA segments. To delimit the boundaries of the diversified DNA, the catalytically dead Cas9 (dCas9) is tethered with custom-designed crRNAs as a "roadblock" for BD-T7RNAP elongation. While the efficiency of this platform is demonstrated in E. coli, the system can be adapted to a variety of bacterial and eukaryotic hosts.