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As an effective programmable DNA targeting tool, CRISPR-Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch type, dDrG (D = A, T, G), only showed moderate impairment on binding. To quantitatively understand the causal relationship between mismatch and binding behaviour of dCas9, we further established a biophysical model, and found that the thermodynamic properties of base-pairing coupled with strand invasion process, to a large extent, can account for the observed mismatch-activity landscape. Finally, we repurposed this model, together with a convolutional neural network constructed based on the same mechanism, as a predictive tool to guide the rational design of sgRNA in bacterial CRISPR interference., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we systematically profiled sgRNA activity by co-expressing a genome-scale library (∼70 000 sgRNAs) with Cas9 or its specificity-improved mutant in Escherichia coli. Based on this large-scale dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables selecting highly active sgRNAs for any locus across the genome in this model organism. We also identified 'resistant' genomic loci with respect to CRISPR/Cas9 activity, notwithstanding the highly accessible DNA in bacterial cells. Moreover, we found that previous sgRNA activity prediction models that were trained on mammalian cell datasets were inadequate when coping with our results, highlighting the key limitations and biases of previous models. We hence developed an integrated algorithm to accurately predict highly effective sgRNAs, aiming to facilitate CRISPR/Cas9-based genome engineering, screenings and antimicrobials design in bacteria. We also isolated the important sgRNA features that contribute to DNA cleavage and characterized their key differences among wild type Cas9 and its mutant, shedding light on the biophysical mechanisms of the CRISPR/Cas9 system.

Aims: Avermectins are major antiparasitic agents used commercially in animal health, agriculture and human infections. To improve the fermentation efficiency of avermectins, for the first time a plasma jet generated by a novel atmospheric pressure glow discharge (APGD) was employed to generate mutations in Streptomyces avermitilis., Methods and Results: The APGD plasma jet, driven by a radio frequency (RF) power supply with water-cooled and bare-metallic electrodes, was used as a new mutation method to treat the spores of S. avermitilis. The plasma jet yielded high total (over 30%) and positive (about 21%) mutation rates on S. avermitilis, and a mutated strain, designated as G1-1 with high productivity of avermectin B1a and genetic stability, was obtained., Conclusions: Because of the low jet temperature, the high concentrations of the chemically reactive species and the flexibility of its operation, the RF APGD plasma jet has a strong mutagenic effect on S. avermitilis., Significance and Impact of the Study: This is a proof-of-concept study for the use of an RF APGD plasma jet for inducing mutations in microbes. We have shown that the RF APGD plasma jet could be developed as a promising and convenient mutation tool for the fermentation industry and for use in biotechnology research.

Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus/Methylocystis, type I methanotrophs related to Methylobacter/Methylosoma and Methylococcus, and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using (13)CH(4) were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella, which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium, were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.

In order to construct an expression system for the particulate methane mono-oxygenase (pMMO) gene (pmo), the structural gene cluster pmoCAB amplified from Methylosinus trichosporium OB3b was inserted into a shuttle vector pBS305 under the control of a dsz promoter and transformed into Rhodococcus erythropolis LSSE8-1. A stable transformant was successfully obtained using ethane as the sole carbon source. Fluorescence in situ hybridization results showed that the dsz promoter allowed the pmo genes to be transcribed in the recombinant strain. The effects of Cu2+ and Zn2+ concentrations on cell growth and pMMO activity in ethane-containing medium were examined. It was discovered that 7.5 microM Cu2+ and 1.8 microM Zn2+ were suitable to achieve high cell concentration and pMMO activity, but the amount of methanol accumulated during methane oxidation by the recombinant strain was still low.

A gfp- and kanamycin-resistance gene-containing plasmid pUCGK was successfully constructed and transformed into Enterobacter aerogenes to develop a rapid GFP-based method for quantifying the bacterial concentration under anaerobic conditions for production of biohydrogen. Since the use of GFP as a molecular reporter is restricted by its requirement for oxygen in the development of the fluorophore, fluorescence detection for the fluorescent E. aerogenes grown anaerobically for hydrogen production was performed by developing a method of aerobic fluorescence recovery (AFR) of the anaerobically expressed GFP. By using this AFR method, rapid and non-disruptive cell quantification of E. aerogenes by fluorescence density was achieved for analyzing the hydrogen production process.