Cysteine-independent CRISPR-Associated Protein Labeling for Presentation and Co-delivery of Molecules Toward Genetic and Epigenetic Regulations.

Labeling of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins (Cas) remains an immense challenge for their genome engineering applications. To date, cysteine-mediated bioconjugation is the most efficient strategy for labeling Cas proteins. However, introducing a cysteine residue in the protein at the right place might be challenging without perturbing the enzymatic activity. We report a method that does not require cysteine residues for small molecule presentation on the CRISPR-associated protein SpCas9 for in vitro protein detection, probing cellular protein expression, and nuclear co-delivery of molecules in mammalian cells. We repurposed a simple protein purification tag His6 peptide for non-covalent labeling of molecules on the CRISPR enzyme SpCas9. The small molecule labeling enabled us to rapidly detect SpCas9 in a biochemical assay. We demonstrate that small molecule labeling can be utilized for probing bacterial protein expression in realtime. Furthermore, we coupled SpCas9's nuclear-targeting ability in co-delivering the presenting small molecules to the mammalian cell nucleus for prospective genome engineering applications. Furthermore, we demonstrate that the method can be generalized to label oligonucleotides for multiplexing CRISPR-based genome editing and template-mediated DNA repair applications. This work paves the way for genomic loci-specific bioactive small molecule and oligonucleotide co-delivery toward genetic and epigenetic regulations.

CRISPR-Based Precision Molecular Diagnostics for Disease Detection and Surveillance.

Sensitive, rapid, and portable molecular diagnostics is the future of disease surveillance, containment, and therapy. The recent SARS-CoV-2 pandemic has reminded us of the vulnerability of lives from ever-evolving pathogens. At the same time, it has provided opportunities to bridge the gap by translating basic molecular biology into therapeutic tools. One such molecular biology technique is CRISPR (clustered regularly interspaced short palindromic repeat) which has revolutionized the field of molecular diagnostics at the need of the hour. The use of CRISPR-Cas systems has been widespread in biology research due to the ease of performing genetic manipulations. In 2012, CRISPR-Cas systems were, for the first time, shown to be reprogrammable, i.e., capable of performing sequence-specific gene editing. This discovery catapulted the field of CRISPR-Cas research and opened many unexplored avenues in the field of gene editing, from basic research to therapeutics. One such field that benefitted greatly from this discovery was molecular diagnostics, as using CRISPR-Cas technologies enabled existing diagnostic methods to become more sensitive, accurate, and portable, a necessity in disease control. This Review aims to capture some of the trajectories and advances made in this arena and provides a comprehensive understanding of the methods and their potential use as point-of-care diagnostics.

In silico structural and functional modelling of Antifreeze protein (AFP) sequences of Ocean pout (Zoarces americanus, Bloch & Schneider 1801).

Antifreeze proteins (AFPs) are known to polypeptide components formed by certain plants, animals, fungi and bacteria which support to survive in sub-zero temperature. Current study highlighted the seven different antifreeze proteins of fish Ocean pout (Zoarces americanus), in which protein (amino acids sequence) were collected from National Centre for Biotechnology Information and finely characterized using several in silico tools. Such biocomputational techniques applied to figure out the physicochemical, functional and conformational characteristics of targeted AFPs. Multiple physicochemical properties such as Isoelectric Point, Extinction Coefficient and Instability Index, Aliphatic Index, Grand Average Hydropathy were calculated and analysed by ExPASy-ProtParam prediction web server. EMBOSS: pepwheel online tool was used to represent the protein sequences in a helical form. The primary structure analysis shows that most of the AFPs are hydrophobic in nature due to the high content of non-polar residues. The secondary structure of these proteins was calculated using SOPMA tool. SOSUI server and CYS_REC program also run for ideal prediction of transmembrane helices and disulfide bridges of experimental proteins respectively. The modelling of 3D structures of seven desired AFPs were executed by the homology modelling programmes; SWISS MODEL and ProSA web server. UCSF Chimera, Antheprot 3D, PyMOL and RAMPAGE were used to visualize and analysis of the structural variation of the predicted protein model. MEGA7.0.9 software used to know the phylogenetic relationship among these AFPs. These models offered excellent and reliable baseline information for functional characterization of the experimentally derived protein domain composition by using the advanced tools and techniques of Computational Biology.