Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering
| Autor: | Christina D. Smolke, Aaron Cravens, Jonathan T. Sockolosky, Deze Kong, Osman K. Jamil |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Mutation rate Antifungal Agents Saccharomyces cerevisiae Proteins Science Mutant General Physics and Astronomy Saccharomyces cerevisiae Computational biology Protein Engineering 010402 general chemistry 01 natural sciences Article General Biochemistry Genetics and Molecular Biology Viral Proteins 03 medical and health sciences Drug Resistance Fungal Escherichia coli medicine T7 RNA polymerase Promoter Regions Genetic Gene Synthetic biology Polymerase Multidisciplinary biology Mutagenesis DNA-Directed RNA Polymerases General Chemistry Protein engineering 0104 chemical sciences Pyrimethamine 030104 developmental biology Genetic engineering Mutation (genetic algorithm) biology.protein Directed Molecular Evolution medicine.drug |
| Zdroj: | Nature Communications, Vol 12, Iss 1, Pp 1-12 (2021) Nature Communications |
| ISSN: | 2041-1723 |
| DOI: | 10.1038/s41467-021-21876-z |
| Popis: | 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. Existing in vivo mutagenesis tools are limited by low mutation diversity and mutation rates. Here the authors present TRIDENT for targeted, continual and inducible diversification of genes of interest using deaminases fused to T7 RNA polymerase. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink