User-defined single pot mutagenesis using unamplified oligo pools

Medina-Cucurella, Angélica V; Steiner, Paul J; Faber, Matthew S.; Beltrán, Jesús; Borelli, Alexandra N.; Kirby, Monica B; Cutler, Sean R.; Whitehead, Timothy A.

doi:10.1093/protein/gzz013

Cited by 27 publications

(42 citation statements)

References 11 publications

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“…If the distribution of variant frequencies in the library were uniform, roughly 4.6-fold coverage would be required for 99% coverage (Bosley et al, 2005). However, variant frequencies with nicking mutagenesis are typically distributed log-normally, which necessitates higher fold coverage (Wrenbeck et al, 2016;Medina-Cucurella et al, 2019). Figure 2 shows simulated coverage results demonstrating that 100-fold coverage is a better target.…”

Section: Discussionmentioning

confidence: 99%

“…At the same time, oligonucleotide pool technology, which yields tens of thousands of specifically designed oligos in one pot, has rapidly advanced. Oligo-pools afford practical and economical benefits to the original NM protocol, permitting many more mutations to be designed at lower cost (Medina-Cucurella et al, 2019). The combination of nicking mutagenesis with oligo pool-derived primers may benefit any research where many amino acid substitutions are desired at one or many sites in a target protein, and specifically in directed evolution studies applicable to protein design and evolutionary biology.…”

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confidence: 99%

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A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis

Steiner¹,

Baumer²,

Whitehead³

2020

BIO-PROTOCOL

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Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking restriction endonuclease and exonuclease treatment. Mutagenic primers encoding the desired mutations are annealed to the resulting circular single-stranded DNA, extended with high-fidelity polymerase, and ligated into covalently closed circular DNA by Taq DNA ligase. The heteroduplex DNA is resolved by selective degradation of the template strand. The complementary strand is synthesized and ligated, resulting in a library of mutated covalently closed circular plasmids. It was later shown that because very little primer is used in the procedure, resuspended oligo pools, which normally require amplification before use, can be used directly in the mutagenesis procedure. Because oligo pools can contain tens of thousands of unique oligos, this enables the construction of libraries of tens of thousands of user-defined mutations in a single-pot mutagenesis reaction, which significantly improves the utility of NM as described below. Use of oligo pools afford an economically advantageous approach to mutagenic experiments. First, oligo pool synthesis is much less expensive per nucleotide synthesized than conventional synthesis. Second, a mixed pool may be generated and used for mutagenesis of multiple different genes. To use the same oligo-pool for mutagenesis of a variety of genes, the user must only quantify the fraction of the oligo-pool specific to her mutagenic experiment and adjust the volume and effective concentration of the oligo-pool for use in nicking mutagenesis.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis

Steiner¹,

Baumer²,

Whitehead³

2020

BIO-PROTOCOL

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show abstract

“…Saturation mutagenesis is the exhaustive examination of amino acid changes within a discrete sequence region. This approach, enabled by efficient chemical synthesis of combinatorial DNA libraries (Medina-Cucurella et al, 2019) or by polymerase chain reaction (PCR) with trimer phosphoramidites (Filipovska, Razif, Nygård, & Rackham, 2011), offers comprehensive and largely unbiased exploration of the sequence space but is limited by practical constraints to targeting short protein sequences.…”

Section: Mutant Library Constructionmentioning

confidence: 99%

Building artificial genetic circuits to understand protein function

Scott

Mathews

Filipovska

et al. 2020

Methods in Enzymology

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Intrinsic protein properties that may not be apparent by only examining three-dimensional structures can be revealed by careful analysis of mutant protein variants. Deep mutational scanning is a technique that allows the functional analysis of millions of protein variants in a single experiment. To enable this high-throughput technique, the mutant genotype of protein variants must be coupled to a selectable function. This chapter outlines how artificial genetic circuits in the yeast Saccharomyces cerevisiae can maintain the genotype-phenotype link, thus enabling the general application of this approach. To do this, we describe how to engineer genetic selections in yeast, methods to construct mutant libraries, and how to analyze sequencing data. We investigate the structure-function relationships of the antimicrobial resistance protein TetX to illustrate this process. In doing so, we demonstrate that deep mutational scanning is a powerful method to dissect the importance of individual residues for the inactivation of antibiotics analogues, with consequences for the rational design of new drugs to combat antimicrobial resistance.

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“…The processed data is available in the supporting information. Additionally, the oligo pool sequences, input files for generating the oligo pool, and an example of the command line input to execute the script from Medina-Cucurella et al (2019) are available in the Supplemental Information associated with this paper.…”

Section: Data Availabilitymentioning

confidence: 99%

“…In NM, oligos encode the desired mutations by mismatch with the parental template. On-chip ink-jet printed oligo pools have been integrated into NM (Medina-Cucurella et al 2019), so one can now construct heterogeneous libraries of oligos that contain tens to hundreds of thousands of high fidelity, unique sequences (Kosuri andChurch 2014, Medina-Cucurella et al 2019) with a low per base pair cost.…”

Section: Introductionmentioning

confidence: 99%

Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly

Faber

Leuven

Ederer

et al. 2019

Preprint

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Here we present a novel protocol for the construction of saturation single-site-and massive multi-site-mutant libraries of a bacteriophage. We segmented the ΦX174 genome into 14 non-toxic and non-replicative fragments compatible with golden gate assembly. We next used nicking mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near-comprehensive single-site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned). Libraries possessed greater than 99% of all 11,860 programmed mutations. Golden Gate cloning was then used to assemble the complete ΦX174 mutant genome and generate libraries of infective viruses. This protocol will enable reverse genetics experiments for studying viral evolution and, with some modifications, can be applied for engineering of therapeutically relevant bacteriophages with larger genomes.

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User-defined single pot mutagenesis using unamplified oligo pools

Cited by 27 publications

References 11 publications

A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis

A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis

Building artificial genetic circuits to understand protein function

Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly

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