Advances in the application of CRISPR-Cas technology in rapid detection of pathogen nucleic acid

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are widely used as gene editing tools in biology, microbiology, and other fields. CRISPR is composed of highly conserved repetitive sequences and spacer sequences in tandem. The spacer sequence has homology with foreign nucleic acids such as viruses and plasmids; Cas effector proteins have endonucleases, and become a hotspot in the field of molecular diagnosis because they recognize and cut specific DNA or RNA sequences. Researchers have developed many diagnostic platforms with high sensitivity, high specificity, and low cost by using Cas proteins (Cas9, Cas12, Cas13, Cas14, etc.) in combination with signal amplification and transformation technologies (fluorescence method, lateral flow technology, etc.), providing a new way for rapid detection of pathogen nucleic acid. This paper introduces the biological mechanism and classification of CRISPR-Cas technology, summarizes the existing rapid detection technology for pathogen nucleic acid based on the trans cleavage activity of Cas, describes its characteristics, functions, and application scenarios, and prospects the future application of this technology

Control of Nucleophile Chemoselectivity in Cyanobactin YcaO Heterocyclases PatD and TruD

YcaO proteins are ubiquitous in living organisms, where they perform crucial post-translational modifications of peptides and proteins. They are used extensively in biotechnology in companies and academic settings around the world. They also underlie some of the most important natural products in pharmaceutical development, such as thiopeptides (thiostrepton, etc.). Here, we solve one of the major outstanding mysteries behind YcaO proteins: how they exert precision selectivity of the nucleophile. The resulting findings have major implications in understanding the >30,000 YcaO proteins currently in sequencing databases and will be used widely for precision synthetic biology applications.</div

Precisely Regulated and Efficient Locking of Linear Peptides into Stable Multicyclic Topologies through a One-Pot Reaction

We report the discovery of a small phenyl molecule with four isosteric thiolate-reactive groups of sequentially varied reactivity. This molecule was exploited in combination with cysteine/penicillamine thiolates of different nucleophilic reactivity for precisely regulated and efficient locking (PROP-locking) of linear peptides into multicyclic topologies through a one-pot reaction. The PROP-locking relies on multistep and sequential thiolate/ fluorine nucleophilic substitutions, which is not only rapid but highly specific, thus enabling rapid locking of peptides with high amino acid diversities without protecting groups. Several tricyclic peptide templates and bioactive peptides were designed and synthesized using the PROPlocking strategy. We believe that tricyclic peptides precisely locked through stable thioether bonds should be promising structurally constrained scaffolds for developing potential therapeutics and target ligands

Orthogonal Cysteine–Penicillamine Disulfide Pairing for Directing the Oxidative Folding of Peptides

Precise disulfide pairing in synthetic peptides usually is achieved using orthogonal protecting group strategies or relies on primary sequence manipulation. Orthogonal disulfide pairing technology should be promising for directing the rational folding of multicyclic peptides from the fully reduced peptides. Here, we report a discovery on the orthogonality between heterodisulfide pairing of cysteine (Cys) and penicillamine (Pen) and formation of Cys-Cys/Pen-Pen homodisulfides. The orthogonal Cys-Pen disulfide pairing can be exploited for highly selective production of certain (multi)­cyclic structures (or even a sole structure without isomers) through direct oxidation in air or thiol–disulfide exchanges in redox media. This strategy makes rational folding of multicyclic peptides without protecting groups, sequence manipulation, and complex synthetic reactions a reality, thus providing invaluable assets to peptide communities, and should greatly benefit the development of multicyclic peptide therapeutics and ligands