Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering

Aaron Cravens, Osman K. Jamil, Deze Kong, Jonathan T. Sockolosky and Christina D. Smolke ()
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Aaron Cravens: 443 Via Ortega, MC 4245, Stanford University
Osman K. Jamil: 443 Via Ortega, MC 4245, Stanford University
Deze Kong: 443 Via Ortega, MC 4245, Stanford University
Jonathan T. Sockolosky: Stanford University School of Medicine
Christina D. Smolke: 443 Via Ortega, MC 4245, Stanford University

Nature Communications, 2021, vol. 12, issue 1, 1-12

Abstract: Abstract Random mutagenesis is a technique used to generate diversity and engineer biological systems. In vivo random mutagenesis generates diversity directly in a host organism, enabling applications such as lineage tracing, continuous evolution, and protein engineering. Here we describe TRIDENT (TaRgeted In vivo Diversification ENabled by T7 RNAP), a platform for targeted, continual, and inducible diversification at genes of interest at mutation rates one-million fold higher than natural genomic error rates. TRIDENT targets mutagenic enzymes to precise genetic loci by fusion to T7 RNA polymerase, resulting in mutation windows following a mutation targeting T7 promoter. Mutational diversity is tuned by DNA repair factors localized to sites of deaminase-driven mutation, enabling sustained mutation of all four DNA nucleotides at rates greater than 10−4 mutations per bp. We show TRIDENT can be applied to routine in vivo mutagenesis applications by evolving a red-shifted fluorescent protein and drug-resistant mutants of an essential enzyme.

Date: 2021
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DOI: 10.1038/s41467-021-21876-z

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