Development of potent in vivo mutagenesis plasmids with broad mutational spectra
By A. Badran, David R. Liu. 2015
195 citations recorded by the source corpus.
Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms. Random DNA mutagenesis provides genetic diversity both in nature and the laboratory. Here, Badran and Liu present a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that surpasses the mutational efficiency and spectra of the most widely used in vivo and in vitromutagenesis methods.
Subfields: Genetics, Molecular Biology
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Citations in the corpus
- Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, positive): “Since dilution occurs faster than cell division but slower than phage replication, only the SP, and not the host cells, can accumulate mutations 17 .”