Sequence modification in copoly(ester-imide)s: a catalytic/supramolecular approach to the evolution and reading of copolymer sequence information

Catalytic ester-interchange reactions, analogous to mutation and recombination, allow new sequence information to be written statistically into poly(ester-imide) chains based on NDI (1,4,5,8-naphthalenetetracarboxylic diimide) units. Thus, both the insertion of the cyclic ester cyclopentadecanolide (“exaltolide”) into an NDI-based homopolymer and quantitative sequence exchange between two different homopoly(ester-imide)s are catalyzed by di-n-butyl tin(IV) oxide. Emerging sequences are identified at the triplet and quintet levels using supramolecular complexation of pyrene-d10 at the NDI residues to amplify the separation of 1H NMR resonances associated with different sequences. In such systems, pyrene is able to act as a “reader molecule” by generating different levels of ring-current shielding from the different patterns of supramolecular binding to all NDI-centered sequences of a given length.

Sequence-Modification in Copoly(ester-Imide)s: A Catalytic/supramolecular Approach to Writing/reading Copolymer Sequence-Information

Catalytic ester-interchange reactions, analogous to mutation and recombination, allow new sequence-information to be written, statistically, into NDI-based poly(ester-imide) chains. Thus, insertion of the cyclic ester cyclopentadecanolide ("exaltolide") into an NDI-based homopolymer, and quantitative sequence-exchange between two different homopoly(ester-imide)s, are catalysed by di-<i>n</i>-butyl tin(IV) oxide. Emerging sequences are identified at the triplet and quintet levels by ¹H NMR analysis, using supramolecular complexation of pyrene-<i>d</i>₁₀ at the NDI residues to amplify the separation of resonances associated with different sequences. In such systems, pyrene is able to act as a "reader-molecule" by generating different levels of ring-current shielding from the different patterns of supramolecular binding to all the NDI-centred sequences of a given length.

Sequence-Modification in Copoly(ester-Imide)s: A Catalytic/supramolecular Approach to Writing/reading Copolymer Sequence-Information

Catalytic ester-interchange reactions, analogous to mutation and recombination, allow new sequence-information to be written, statistically, into NDI-based poly(ester-imide) chains. Thus, insertion of the cyclic ester cyclopentadecanolide ("exaltolide") into an NDI-based homopolymer, and quantitative sequence-exchange between two different homopoly(ester-imide)s, are catalysed by di-<i>n</i>-butyl tin(IV) oxide. Emerging sequences are identified at the triplet and quintet levels by ¹H NMR analysis, using supramolecular complexation of pyrene-<i>d</i>₁₀ at the NDI residues to amplify the separation of resonances associated with different sequences. In such systems, pyrene is able to act as a "reader-molecule" by generating different levels of ring-current shielding from the different patterns of supramolecular binding to all the NDI-centred sequences of a given length.

Streamlined, recombinase-free genome editing with CRISPR-Cas9 inLactobacillus plantarumreveals barriers to efficient editing

ABSTRACTLactic-acid bacteria such asLactobacillus plantarumare commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are currently being employed to elucidate and enhance these microbes’ beneficial properties. The most advanced tools to-date require heterologous single-stranded DNA recombinases to integrate short oligonucleotides followed by using CRISPR-Cas9 to eliminate cells harboring unedited sequences. Here, we show that encoding the recombineering template on a replicating plasmid allowed efficient genome editing with CRISPR-Cas9 in multipleL. plantarumstrains without a recombinase. This strategy accelerated the genome-editing pipeline and could efficiently introduce a stop codon inribB, silent mutations inackA, and a complete deletion oflacM. In contrast, oligo-mediated recombineering with CRISPR-Cas9 proved far less efficient in at least one instance. We also observed unexpected outcomes of our recombinase-free method, including an ~1.3-kb genomic deletion when targetingribBin one strain, and reversion of a point mutation in the recombineering template in another strain. Our method therefore can streamline targeted genome editing in different strains ofL. plantarum, although the best means of achieving efficient editing may vary based on the selected sequence modification, gene, and strain.