In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9

Beatriz Álvarez, Mario Mencía, Víctor Lorenzo and Luis Ángel Fernández ()
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Beatriz Álvarez: Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Darwin 3, Campus UAM Cantoblanco
Mario Mencía: Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas – Universidad Autónoma de Madrid), Nicolas Cabrera 1, Campus UAM Cantoblanco
Víctor Lorenzo: Systems Biology Program, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Darwin 3, Campus UAM Cantoblanco
Luis Ángel Fernández: Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Darwin 3, Campus UAM Cantoblanco

Nature Communications, 2020, vol. 11, issue 1, 1-14

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-20230-z

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