Abstract
In vivo mutagenesis systems accelerate directed protein evolution but often show restricted capabilities and deleterious off-site mutations on cells. To overcome these limitations, here we report 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. 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. Using this T7-targeted dCas9-limited in vivo mutagenesis (T7-DIVA) system, rapid molecular evolution of the antibiotic resistance gene TEM-1 is achieved. While the efficiency is demonstrated in E. coli, the system can be adapted to a variety of bacterial and eukaryotic hosts.
Highlights
In vivo mutagenesis systems accelerate directed protein evolution but often show restricted capabilities and deleterious off-site mutations on cells
We report a strategy that fulfill these criteria enabling in vivo mutagenesis of a target gene, using different highly processive protein fusions of base deaminases (BD) and the bacteriophage T7 RNA polymerase (T7RNAP)
We describe the mutagenic action of different BDs (i.e., AID, rAPOBEC1, pmCDA1, and TadA*) fused to T7RNAP on a target genomic DNA segment in E. coli chromosome
Summary
In vivo mutagenesis systems accelerate directed protein evolution but often show restricted capabilities and deleterious off-site mutations on cells To overcome these limitations, here we report 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. To delimit the boundaries of the diversified DNA, the catalytically dead Cas[9] (dCas9) is tethered with custom-designed crRNAs as a “roadblock” for BD-T7RNAP elongation Using this T7-targeted dCas9-limited in vivo mutagenesis (T7-DIVA) system, rapid molecular evolution of the antibiotic resistance gene TEM-1 is achieved. In vitro mutagenesis techniques quickly produce large number of variants of the target gene, but their selection requires, in most cases, cloning and transformation into a host cell for expression. Expression of CDs, such as human AID (activation-induced cytidine deaminase), and orthologs from rat (rAPOBEC1, rat apolipoprotein B mRNA editing enzyme 1) and lamprey
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