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Plasmid-based one-pot saturation mutagenesis

Nature Methods volume 13, pages 928–930 (2016)Cite this article

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Abstract

Deep mutational scanning is a foundational tool for addressing the functional consequences of large numbers of mutants, but a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Here we present nicking mutagenesis, a robust, single-day, one-pot saturation mutagenesis method performed on routinely prepped plasmid dsDNA. The method can be used to produce comprehensive or single- or multi-site saturation mutagenesis libraries.

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Figure 1: Comprehensive single-site nicking mutagenesis.

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Acknowledgements

We thank M. Ostermeier (John Hopkins University) for the oligo sets used in creation of the bla libraries. This research was partially supported by a fellowship from Michigan State University under the Training Program in Plant Biotechnology for Health and Sustainability (T32-GM110523 to E.E.W.), a Howard Hughes Medical Institute Gilliam fellowship (to A.A.), US Department of Agriculture National Institute of Food and Agriculture award 2016-67011-24701 (to J.R.K.), and the US National Science Foundation Career Award 1254238 CBET (to T.A.W.).

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Authors and Affiliations

  1. Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, USA

    Emily E Wrenbeck, James A Stapleton & Timothy A Whitehead

  2. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA

    Justin R Klesmith

  3. Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, USA

    Adebola Adeniran & Keith E J Tyo

  4. Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, Michigan, USA

    Timothy A Whitehead

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  1. Emily E Wrenbeck
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  2. Justin R Klesmith
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  4. Adebola Adeniran
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  5. Keith E J Tyo
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  6. Timothy A Whitehead
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Contributions

E.E.W. and T.A.W. wrote the manuscript with input from all coauthors; E.E.W., T.A.W., J.R.K., and J.A.S. conceived the method; E.E.W., J.R.K., J.A.S., and T.A.W. devised experiments; E.E.W., J.R.K., and A.A. performed experiments; K.E.J.T. and A.A. tested the method.

Corresponding author

Correspondence to Timothy A Whitehead.

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Competing interests

E.E.W., J.R.K., J.A.S. and T.A.W. have filed a provisional patent application covering this method (US provisional number 62/380,717).

Integrated supplementary information

Supplementary Figure 1 Gel snapshots along the optimized nicking mutagenesis method.

Plasmid dsDNA and ssDNA (prepared from bacteriophage) of pEDA5_GFPmut3 are included for size reference. NR = nicking reaction; 2 μg of pEDA5_GFPmut3_Y66H was placed in a 20 μL reaction with 10 U Nt.BbvCI in 1X CutSmart buffer. TP = template preparation; a reaction was ceased after the template preparation phase. MS = mutant strand; a reaction was ceased after the synthesis of the mutant strands, where regeneration of relaxed dsDNA can be seen. 1 kb Plus Ladder (Thermo Fischer Scientific, lane 1) included for size reference. Gel image has been cropped to size.

Supplementary Figure 2 Probability distribution of mutation counts in amiE comprehensive nicking mutagenesis libraries.

Dashed vertical lines represent median (red) and mean (blue) library member read coverage. Panel a shows distribution for reaction 1 and panel b shows the distribution for reaction 2.

Source data

Supplementary Figure 3 Comparison of the probability distributions of site-saturation mutagenesis libraries resulting from nicking mutagenesis or PFunkel mutagenesis.

Because the depth of sequencing coverage varied between the three methods, all samples were normalized to a 200-fold depth of coverage of possible single non-synonymous mutations. The expected library diversity is 820 for Kowalsky et al.1,2 and 1420 for amiE reaction 1 & reaction 2 (this work). a. Cumulative distribution function for the three libraries as a function of normalized sequencing counts. 91.7%, 93.2%, and 97.8% of the library is represented above a threshold of 10 sequencing counts for PFunkel library, amiE reaction 1, and the amiE reaction 2 libraries, respectively. b. Frequency is plotted as a function of sequencing counts for the same three libraries. The experimental data are plotted as symbols, with lines representing a best fit of the data using a log-normal distribution (PFunkel: μ=2, σ=0.49, amiE reaction 1: m=2, σ=0.50. amiE reaction 2: m=2, σ=0.44).

Source data

Supplementary Figure 4 Off-target mutational analysis of amiE input plasmid and mutational libraries by shotgun sequencing.

a-c. Percent mutant allele at each position in the plasmid sequence for the input plasmid (a) amiE reaction 1 library (b) and amiE reaction 2 library (c). Shotgun sequencing reads were aligned to the pEDA3_amiE plasmid using BWA aligner3,4 and the frequency of each base at each position was counted using bam-readcount (www.github.com). Percent mutant allele was calculated for each position by summing all non-wildtype allele counts and diving by total reads at that position. Overlain red curves indicate depth of sequencing coverage at each position. d-e. Background subtracted percent mutant allele for each position in plasmid sequence of amiE reaction 1 library (d) and amiE reaction 2 library (e).

Source data

Supplementary Figure 5 bla library coverage distributions.

Probability distribution of mutation counts in bla comprehensive nicking mutagenesis libraries. Dashed vertical lines represent median (red) and mean (blue) library member read coverage. b. Cumulative distribution function for the three libraries as a function of normalized sequencing counts.

Source data

Supplementary Figure 6 Schematic overview of single- or multi-site nicking mutagenesis.

After the preparation of an ssDNA template, an annealing reaction is set up with a single or mixed set of mutagenic oligos at a 5:1 primer:template ratio (for each oligo). Next, reagents and enzymes necessary to synthesize the mutant strands are added. The remainder of the protocol is identical to comprehensive nicking mutagenesis.

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Wrenbeck, E., Klesmith, J., Stapleton, J. et al. Plasmid-based one-pot saturation mutagenesis. Nat Methods 13, 928–930 (2016). https://doi.org/10.1038/nmeth.4029

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