CRISPR-Cas9-assisted genome editing in E. coli elevates the frequency of unintended mutations.

Cas-assisted lambda Red recombineering techniques have rapidly become a mainstay of bacterial genome editing. Such techniques have been used to construct both individual mutants and massive libraries to assess the effects of genomic changes. We have found that a commonly used Cas9-assisted editing method results in unintended mutations elsewhere in the genome in 26% of edited clones. The unintended mutations are frequently found over 200 kb from the intended edit site and even over 10 kb from potential off-target sites. We attribute the high frequency of unintended mutations to error-prone polymerases expressed in response to dsDNA breaks introduced at the edit site. Most unintended mutations occur in regulatory or coding regions and thus may have phenotypic effects. Our findings highlight the risks associated with genome editing techniques involving dsDNA breaks in E. coli and likely other bacteria and emphasize the importance of sequencing the genomes of edited cells to ensure the absence of unintended mutations.

Synonymous edits in the Escherichia coli genome have substantial and condition-dependent effects on fitness.

CRISPR-Cas-based genome editing is widely used in bacteria at scales ranging from construction of individual mutants to massively parallel libraries. This procedure relies on guide RNA-directed cleavage of the genome followed by repair with a template that introduces a desired mutation along with synonymous "immunizing" mutations to prevent re-cleavage of the genome after editing. Because the immunizing mutations do not change the protein sequence, they are often assumed to be neutral. However, synonymous mutations can change mRNA structures in ways that alter levels of the encoded proteins. We have tested the assumption that immunizing mutations are neutral by constructing a library of over 50,000 edits that consist of only synonymous mutations in Escherichia coli. Thousands of edits had substantial effects on fitness during growth of E. coli on acetate, a poor carbon source that is toxic at high concentrations. The percentage of high-impact edits varied considerably between genes and at different positions within genes. We reconstructed clones with high-impact edits and found that 69% indeed had significant effects on growth in acetate. Interestingly, fewer edits affected fitness during growth in glucose, a preferred carbon source, suggesting that changes in protein expression caused by synonymous mutations may be most important when an organism encounters challenging conditions. Finally, we showed that synonymous edits can have widespread effects; a synonymous edit at the 5' end of ptsI altered expression of hundreds of genes. Our results suggest that the synonymous immunizing edits introduced during CRISPR-Cas-based genome editing should not be assumed to be innocuous.

How to Recruit a Promiscuous Enzyme to Serve a New Function.

Promiscuous enzymes can be recruited to serve new functions when a genetic or environmental change makes catalysis of a novel reaction important for fitness or even survival. Subsequently, gene duplication and divergence can lead to evolution of an efficient and specialized new enzyme. Every organism likely has thousands of promiscuous enzyme activities that provide a vast reservoir of catalytic potential. However, much of this potential may not be accessible. We compiled kinetic parameters for promiscuous reactions catalyzed by 108 enzymes. The median value of kcat/KM is a very modest 31 M-1 s-1. Based upon the fluxes through metabolic pathways in E. coli, we estimate that many, if not most, promiscuous activities are too inefficient to impact fitness. However, mutations can elevate the level of an insufficient promiscuous activity by increasing enzyme expression, improving kcat/KM, or altering concentrations of the promiscuous and native substrates and allosteric regulators. Particularly in large bacterial populations, stochastic mutations may provide a viable pathway for recruitment of even inefficient promiscuous activities.

Determinants of sulphur chemolithoautotrophy in the extremely thermoacidophilic Sulfolobales.

Species in the archaeal order Sulfolobales thrive in hot acid and exhibit remarkable metabolic diversity. Some species are chemolithoautotrophic, obtaining energy through the oxidation of inorganic substrates, sulphur in particular, and acquiring carbon through the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) CO2 -fixation cycle. The current model for sulphur oxidation in the Sulfolobales is based on the biochemical analysis of specific proteins from Acidianus ambivalens, including sulphur oxygenase reductase (SOR) that disproportionates S° into H2 S and sulphite (SO3 2- ). Initial studies indicated SOR catalyses the essential first step in oxidation of elemental sulphur, but an ancillary role for SOR as a 'recycle' enzyme has also been proposed. Here, heterologous expression of both SOR and membrane-bound thiosulphate-quinone oxidoreductase (TQO) from Sulfolobus tokodaii 'restored' sulphur oxidation capacity in Sulfolobus acidocaldarius DSM639, but not autotrophy, although earlier reports indicate this strain was once capable of chemolithoautotrophy. Comparative transcriptomic analyses of Acidianus brierleyi, a chemolithoautotrophic sulphur oxidizer, and S. acidocaldarius DSM639 showed that while both share a strong transcriptional response to elemental sulphur, S. acidocaldarius DSM639 failed to upregulate key 3-HP/4-HB cycle genes used by A. brierleyi to drive chemolithoautotrophy. Thus, the inability for S. acidocaldarius DSM639 to grow chemolithoautotrophically may be rooted more in gene regulation than the biochemical capacity.