HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice

The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets

Import of Aspartate and Malate by DcuABC Drives H2/Fumarate Respiration to Promote Initial Salmonella Gut-Lumen Colonization in Mice

ISSN:1931-3128ISSN:1934-606

Real-Time Metabolomics on Living Microorganisms Using Ambient Electrospray Ionization Flow-Probe

Microorganisms such as bacteria and fungi produce a variety of specialized metabolites that are invaluable for agriculture, biological research, and drug discovery. However, the screening of microbial metabolic output is usually a time intensive task. Here we utilize a liquid micro-junction surface sampling probe for electrospray ionization mass spectrometry to extract and ionize metabolite mixtures directly from living microbial colonies grown on soft nutrient agar in Petri-dishes without any sample pre-treatment. To demonstrate the method is robust, this technique was applied to observe the metabolic output of more than 30 microorganisms, including yeast, filamentous fungi, pathogens, and marine-derived bacteria, that were collected worldwide. Diverse natural products produced from different microbes, including Streptomyces coelicolor, Bacillus subtilis, and Pseudomonas aeruginosa are further characterized