A Processive Protein Chimera Introduces Mutations across Defined DNA Regions In Vivo.

Laboratory time scale evolution in vivo relies on the generation of large, mutationally diverse gene libraries to rapidly explore biomolecule sequence landscapes. Traditional global mutagenesis methods are problematic because they introduce many off-target mutations that are often lethal and can engender false positives. We report the development and application of the MutaT7 chimera, a potent and highly targeted in vivo mutagenesis agent. MutaT7 utilizes a DNA-damaging cytidine deaminase fused to a processive RNA polymerase to continuously direct mutations to specific, well-defined DNA regions of any relevant length. MutaT7 thus provides a mechanism for in vivo targeted mutagenesis across multi-kb DNA sequences. MutaT7 should prove useful in diverse organisms, opening the door to new types of in vivo evolution experiments.

[1]  R. Kohli,et al.  Harnessing natural DNA modifying activities for editing of the genome and epigenome. , 2018, Current opinion in chemical biology.

[2]  Rick L. Stevens,et al.  A communal catalogue reveals Earth’s multiscale microbial diversity , 2017, Nature.

[3]  G. K. Sandve,et al.  Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1 , 2017, Scientific Reports.

[4]  Kevin T. Zhao,et al.  Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity , 2017, Science Advances.

[5]  Daesik Kim,et al.  Genome-wide target specificities of CRISPR RNA-guided programmable deaminases , 2017, Nature Biotechnology.

[6]  Gaelen T. Hess,et al.  Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells , 2016, Nature Methods.

[7]  Hal S Alper,et al.  In vivo continuous evolution of genes and pathways in yeast , 2016, Nature Communications.

[8]  Yan Song,et al.  Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells , 2016, Nature Methods.

[9]  A. Kondo,et al.  Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems , 2016, Science.

[10]  D. G. Gibson,et al.  Vibrio natriegens as a fast-growing host for molecular biology , 2016, Nature Methods.

[11]  Vitor B. Pinheiro,et al.  Selection platforms for directed evolution in synthetic biology , 2016, Biochemical Society transactions.

[12]  Goran Jovanovic,et al.  Single-step method for β-galactosidase assays in Escherichia coli using a 96-well microplate reader , 2016, Analytical biochemistry.

[13]  David R. Liu,et al.  Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage , 2016, Nature.

[14]  David R. Liu,et al.  Development of potent in vivo mutagenesis plasmids with broad mutational spectra , 2015, Nature Communications.

[15]  Arjun Ravikumar,et al.  An orthogonal DNA replication system in yeast. , 2014, Nature chemical biology.

[16]  Merja Penttilä,et al.  Yeast oligo-mediated genome engineering (YOGE). , 2013, ACS synthetic biology.

[17]  David M. Simcha,et al.  Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication , 2011, Nucleic acids research.

[18]  J. A. Ruiz-Masó,et al.  Protein p56 from the Bacillus subtilis phage ϕ29 inhibits DNA-binding ability of uracil-DNA glycosylase , 2007, Nucleic acids research.

[19]  J. W. Campbell,et al.  Experimental Determination and System Level Analysis of Essential Genes in Escherichia coli MG1655 , 2003, Journal of bacteriology.

[20]  O. Berg,et al.  Biological Costs and Mechanisms of Fosfomycin Resistance in Escherichia coli , 2003, Antimicrobial Agents and Chemotherapy.

[21]  Manel Camps,et al.  Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Jeffrey H. Miller,et al.  Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome. , 2003, DNA repair.

[23]  M. Nussenzweig,et al.  Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand , 2003, Nature Immunology.

[24]  M. Neuberger,et al.  AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification , 2002, Nature.

[25]  Dimitrios Panagopoulos,et al.  A mutation in the folA promoter delays adaptation to minimal medium by Escherichia coli K‐12 , 2002, Journal of basic microbiology.

[26]  M. Rosbash,et al.  T7 RNA polymerase-directed transcripts are processed in yeast and link 3' end formation to mRNA nuclear export. , 2002, RNA.

[27]  V. Thiel,et al.  Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. , 2001, The Journal of general virology.

[28]  T. Lindahl,et al.  Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication. , 2000, Molecular cell.

[29]  W. Mcallister,et al.  Promoter specificity determinants of T7 RNA polymerase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[30]  James Scott,et al.  Evolutionary origins of apoB mRNA editing: Catalysis by a cytidine deaminase that has acquired a novel RNA-binding motif at its active site , 1995, Cell.

[31]  D. Stalker,et al.  Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[32]  V. Sandig,et al.  A mutant T7 phage promoter is specifically transcribed by T7-RNA polymerase in mammalian cells. , 1993, European journal of biochemistry.

[33]  J. Miller,et al.  A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[34]  F. Goldstein,et al.  Genetic aspects and epidemiologic implications of resistance to trimethoprim. , 1982, Reviews of infectious diseases.

[35]  I. Tessman,et al.  Mutagenic Effects of Hydroxylamine in vivo , 1965, Science.

[36]  A. Greener,et al.  An efficient random mutagenesis technique using an E. coli mutator strain. , 1996, Methods in molecular biology.