This review reports the recent advances in surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) platforms for the diagnosis of infectious diseases. As observed through the recent infection outbreaks of COVID-19 worldwide, a timely diagnosis of the disease is critical for preventing the spread of a disease and to ensure epidemic preparedness. In this regard, an innovative point-of-care diagnostic method is essential. Recently, SERS-based assay platforms have received increasing attention in medical communities owing to their high sensitivity and multiplex detection capability. In contrast, LFAs provide a user-friendly and easily accessible sensing platform. Thus, the combination of LFAs with a SERS detection system provides a new diagnostic modality for accurate and rapid diagnoses of infectious diseases. In this context, we briefly discuss the recent application of LFA platforms for the POC diagnosis of SARS-CoV-2. Thereafter, we focus on the recent advances in SERS-based LFA platforms for the early diagnosis of infectious diseases and their applicability for the rapid diagnosis of SARS-CoV-2. Finally, the key issues that need to be addressed to accelerate the clinical translation of SERS-based LFA platforms from the research laboratory to the bedside are discussed.

T cells are critical to fight pathogenic microbes and combat malignantly transformed cells in the fight against cancer. To exert their effector function, T cells produce effector molecules, such as the pro-inflammatory cytokines IFN-γ, TNF-α and IL-2. Tumors possess many inhibitory mechanisms that dampen T cell effector function, limiting the secretion of cytotoxic molecules. As a result, the control and elimination of tumors is impaired. Through recent advances in genomic editing, T cells can now be successfully modified via CRISPR/Cas9 technology. For instance, engaging (post-)transcriptional mechanisms to enhance T cell cytokine production, the retargeting of T cell antigen specificity or rendering T cells refractive to inhibitory receptor signaling can augment T cell effector function. Therefore, CRISPR/Cas9-mediated genome editing might provide novel strategies for cancer immunotherapy. Recently, the first-in-patient clinical trial was successfully performed with CRISPR/Cas9-modified human T cell therapy. In this review, a brief overview of currently available techniques is provided, and recent advances in T cell genomic engineering for the enhancement of T cell effector function for therapeutic purposes are discussed.

Revealing the landscape of epigenetic changes in cells during differentiation is important for understanding the development of organisms. In this study, to infer such epigenetic changes during human hematopoiesis, ancestral state estimation based on a phylogenetic tree was applied to map the epigenomic changes in six kinds of histone modifications onto the hierarchical cell differentiation process of hematopoiesis using epigenomes of eight types of differentiated hematopoietic cells. The histone modification changes inferred during hematopoiesis showed that changes that occurred on the branches separating different cell types reflected the characteristics of hematopoiesis in terms of genomic position and gene function. These results suggested that ancestral state estimation based on phylogenetic analysis of histone modifications in differentiated hematopoietic cells could reconstruct an appropriate landscape of histone modification changes during hematopoiesis. Since integration of the inferred changes of different histone modifications could reveal genes with specific histone marks such as active histone marks and bivalent histone marks on each internal branch of cell-type trees, this approach could provide valuable information for understanding the cell differentiation steps of each cell lineage.

Antibiotic resistance in pathogens is a growing threat to human health. Of particular concern is resistance to carbapenem, which is an antimicrobial agent listed as critically important by the World Health Organization. With the global spread of carbapenem-resistant organisms, there is an urgent need for new treatment options. Shewanella algae is an emerging pathogen found in marine environments throughout the world that has increasing resistance to carbapenem. The organism is also a possible antibiotic resistance reservoir in humans and in its natural habitat. The development of CRISPR/Cas9-based methods has enabled precise genetic manipulation. A number of attempts have been made to knock out resistance genes in various organisms. The study used a single plasmid containing CRISPR/Cas9 and recE/recT recombinase to reverse an antibiotic-resistant phenotype in S. algae and showed bla OXA-55 -like gene is essential for the carbapenem resistance. This result demonstrates a potential validation strategy for functional genome annotation in S. algae.

Alport syndrome (AS) is an inherited disorder characterized by glomerular basement membrane (GBM) abnormality and development of chronic kidney disease at an early age. The cause of AS is a genetic mutation in type IV collagen, and more than 80% of patients have X-linked AS (XLAS) with mutation in COL4A5. Although the causal gene has been identified, mechanisms of progression have not been elucidated, and no effective treatment has been developed. In this study, we generated a Col4a5 mutant mouse harboring a nonsense mutation (R471X) obtained from a patient with XLAS using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system. Col4a5 mRNA and protein expressions were not observed in the kidneys of hemizygous R471X male mice. R471X mice showed proteinuria and hematuria. Pathology revealed progression of glomerulosclerosis and interstitial fibrosis by age. Electron microscopy identified irregular thickening in GBM accompanied by irregular lamination. These observations were consistent with the clinical and pathological features of patients with AS and other established models. In addition, our mice models develop end-stage renal disease at the median age of 28 weeks, much later compared to previous models much more consistent with clinical course of human XLAS. Our models have advantages for future experiments in regard with treatment for human XLAS.

α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER \"knock-in\" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreERT2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing.
We utilized CRISPR-Cas9 technology to insert an IRES-CreERT2 sequence into the 3\' UTR of the Glucagon (Gcg) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreERT2 mice. Recombination efficiency in GCG+ pancreatic α-cells and glucagon-like peptide 1 positive (GLP1+) enteroendocrine L-cells was measured in Gcg-CreERT2 ;Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood.
Tamoxifen injection of Gcg-CreERT2 ;Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreERT2 allele were phenotypically normal.
We successfully derived a Gcg-CreERT2 mouse line that expresses CreERT2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.

Gene expression is often controlled by transcriptional repressors during development. Many transcription factors lack intrinsic repressive activity but recruit co-factors that inhibit productive transcription. Here we discuss new insights and models for repression mediated by the Groucho/Transducin-Like Enhancer of split (Gro/TLE) family of co-repressor proteins.

CRISPR/Cas9 technology is a highly promising genome editing tool in the mouse, potentially overcoming the costs and time required for more traditional gene targeting methods in embryonic stem (ES) cells. Recently, compared to the wildtype nuclease, paired Cas9 nickase (Cas9n) combined with single guide RNA (sgRNA) molecules has been found to enhance the specificity of genome editing while reducing off-target effects. Paired Cas9n has been shown to be as efficient as Cas9 for generating insertion and deletion (indel) mutations by non-homologous end joining and targeted deletion in the genome. However, an efficient and reliable approach to the insertion of loxP sites flanking critical exon(s) to create a conditional allele of a target gene remains an elusive goal. In this study, we microinjected Cas9n RNA with sgRNAs together with a single DNA template encoding two loxP sites flanking (floxing) exon 2 of the isoprenoid synthase containing domain (Ispd) into the pronucleus and cytoplasm of C57BL/6NCr one-cell stage zygotes. After surgical transfer, one F0 mouse expressing a conditional allele was produced (at a frequency of ∼8% of live pups born). The floxed allele was transmitted through the germline to F1 progeny, and could be successfully recombined using Cre recombinase. This study indicates that conditional targeting can be accomplished effectively using paired Cas9n and a single DNA template.