Journal
NATURE COMMUNICATIONS
Volume 11, Issue 1, Pages -Publisher
NATURE RESEARCH
DOI: 10.1038/s41467-020-20230-z
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Funding
- Agencia Espanola de Investigacion AEI/MICIU/FEDER, EU [BIO2017-89081-R]
- European Research Council,EU [ERC-2012-ADG_20120314]
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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. In vivo mutagenesis systems can often show restricted capabilities and deleterious off-site mutations. Here the authors fuse base deaminases to T7 RNA polymerase to mutate a target sequence and use dCas9 to define the boundaries of the diversified DNA.
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