Standardized Iterative Genome Editing Method for Escherichia coli Based on CRISPR-Cas9.

The introduction of complex biosynthetic pathways into the hosts' chromosomes is gaining attention with the development of synthetic biology. While CRISPR-Cas9 has been widely employed for gene knock-in, the process of multigene insertion remains cumbersome due to laborious and empirical gene cloning procedures. To address this, we devised a standardized iterative genome editing system for Escherichia coli, harnessing the power of CRISPR-Cas9 and MetClo assembly. This comprehensive toolkit comprises two fundamental elements based on the Golden Gate standard for modular assembly of sgRNA or CRISPR arrays and donor DNAs. We achieved a gene insertion efficiency of up to 100%, targeting a single locus. Expression of tracrRNA using a strong promoter enhances multiplex genomic insertion efficiency to 7.3%, compared with 0.76% when a native promoter is used. To demonstrate the robust capabilities of this genome editing toolbox, we successfully integrated 5-10 genes from the coenzyme B12 biosynthetic pathway ranging from 5.3 to 8 Kb in length into the chromosome of E. coli chassis cells, resulting in 14 antibiotic-free, plasmid-free producers. Following an extensive screening process involving genes from diverse sources, cistronic design modifications, and chromosome repositioning, we obtained a recombinant strain yielding 1.49 mg L-1 coenzyme B12, the highest known titer achieved by using E. coli as the producer. Illuminating its user-friendliness, this genome editing system is an exceedingly versatile tool for expediently integrating complex biosynthetic pathway genes into hosts' genomes, thus facilitating pathway optimization for chemical production.

Author Correction: Nuclei multiplexing with barcoded antibodies for single-nucleus genomics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nuclei multiplexing with barcoded antibodies for single-nucleus genomics.

Single-nucleus RNA-seq (snRNA-seq) enables the interrogation of cellular states in complex tissues that are challenging to dissociate or are frozen, and opens the way to human genetics studies, clinical trials, and precise cell atlases of large organs. However, such applications are currently limited by batch effects, processing, and costs. Here, we present an approach for multiplexing snRNA-seq, using sample-barcoded antibodies to uniquely label nuclei from distinct samples. Comparing human brain cortex samples profiled with or without hashing antibodies, we demonstrate that nucleus hashing does not significantly alter recovered profiles. We develop DemuxEM, a computational tool that detects inter-sample multiplets and assigns singlets to their sample of origin, and validate its accuracy using sex-specific gene expression, species-mixing and natural genetic variation. Our approach will facilitate tissue atlases of isogenic model organisms or from multiple biopsies or longitudinal samples of one donor, and large-scale perturbation screens.

Characterization of Streptococcus pluranimalium from a cattle with mastitis by whole genome sequencing and functional validation.

BACKGROUND: Streptococcus pluranimalium is a new member of the Streptococcus genus isolated from multiple different animal hosts. It has been identified as a pathogen associated with subclinical mastitis, valvular endocarditis and septicaemia in animals. Moreover, this bacterium has emerged as a new pathogen for human infective endocarditis and brain abscess. However, the patho-biological properties of S. pluranimalium remain virtually unknown. The aim of this study was to determine the complete genome sequence of S. pluranimalium strain TH11417 isolated from a cattle with mastitis, and to characterize its antimicrobial resistance, virulence, and carbon catabolism. RESULTS: The genome of S. pluranimalium TH11417, determined by single-molecule real-time (SMRT) sequencing, consists of 2,065,522 base pair (bp) with a G + C content of 38.65%, 2,007 predicted coding sequence (CDS), 58 transfer RNA (tRNA) genes and five ribosome RNA (rRNA) operons. It contains a novel ISSpl1 element (a memeber of the IS3 family) and a Ф11417.1 prophage that carries the mef(A), msr(D) and lnu(C) genes. Consistently, our antimicrobial susceptibility test confirmed that S. pluranimalium TH11417 was resistant to erythromycin and lincomycin. However, this strain did not show virulence in murine pneumonia (intranasal inoculation, 107 colony forming unit - CFU) and sepsis (intraperitoneal inoculation, 107 CFU) models. Additionally, this strain is able to grow with glucose, lactose or galactose as the sole carbon source, and possesses a lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS). CONCLUSIONS: We reported the first whole genome sequence of S. pluranimalium isolated from a cattle with mastitis. It harbors a prophage carrying the mef(A), msr(D) and lnu(C) genes, and is avirulent in the murine infection model.

Characterization of an rmtB-carrying IncI1 ST136 plasmid in avian Escherichia coli isolates from chickens.

The rmtB gene, one of the 16S rRNA methylase genes whose products confer high-level resistance to aminoglycosides, is most prevalent among Enterobacteriaceae strains. In this study, eight non-duplicate rmtB-carrying avian Escherichia coli strains from a farm in China were isolated and characterized, and further examined by phylogenetic grouping, conjugation experiments and PCR-based replicon typing. In addition, the genetic environment of rmtB was investigated by cloning and sequencing. Six rmtB-carrying E. coli were identified as phylogroup A, sequence type (ST) 156 (A-ST156), with two assigned to D-ST117; however, all of them carried the same IncI1 ST136 plasmid. The genetic environment of the rmtB gene in these eight plasmids was the same, as shown by PCR mapping. A multidrug-resistant region carrying blaTEM-1, rmtB, a class 1 integron cassette array (intI1-dfrA12-orfF-aadA2-qacEΔ1-sul1) and aacC2 was characterized on the conjugative IncI1 ST136 plasmid. Co-location of the rmtB gene with a class 1 integron cassette array and aacC2 on the conjugative plasmid will facilitate its maintenance and dissemination.

Myocardial effects of ethanol consumption in the rat with streptozotocin-induced diabetes.

BACKGROUND: Rats with streptozotocin (STZ)-induced diabetes exhibit alterations in cardiac function, ventricular remodeling, and changes in cell signaling, which includes protein kinase C (PKC) isoforms. Moderate consumption of ethanol has a beneficial effect on cardiovascular outcomes in the general population, an effect that has recently been found to extend to patients with diabetes mellitus. We studied the effect of low-dose ethanol consumption on cardiac function, geometry, and PKC isoforms in the rat with STZ-induced diabetes. METHODS: Four groups of rats were studied over 8 to 10 weeks: control, STZ-induced diabetes, 12% (v/v) ethanol consumption, and STZ-induced diabetes plus 4% (v/v) ethanol consumption. Invasive hemodynamic measurements were performed; myocardial tissue was obtained for analysis for total PKC and cytosolic and membrane protein content of PKC-alpha, PKC-delta, and PKC-epsilon, and two-dimensional and M-mode echocardiograms were obtained. RESULTS: Compared with rats with diabetes alone, consumption of 4% ethanol prevented the decrease in left ventricular dP/dt seen with diabetes alone, as well as the increase in left ventricular internal dimension. Up-regulation of PKC-alpha, -delta, and -epsilon occurring in the diabetic animals was also prevented by ethanol consumption, whereas ethanol alone had no effect on PKC isoform pattern. CONCLUSIONS: These data suggest that STZ-induced cardiac remodeling and dysfunction are associated with increases in PKC activity, particularly PKC-alpha, -delta, and -epsilon, and that consumption of ethanol can prevent these changes.