Gene editing that makes target gene modification in the genome by deletion or addition has revolutionized the era of biomedicine. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 emerged as a substantial tool due to its simplicity in use, less cost and extraordinary efficiency than the conventional gene-editing tools, including zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs). However, potential off-target activities are crucial shortcomings in the CRISPR system. Numerous types of approaches have been developed to reduce off-target effects. Here, we review several latest approaches to reduce the off-target effects, including biased or unbiased off-target detection, cytosine or adenine base editors, prime editing, dCas9, Cas9 paired nickase, ribonucleoprotein (RNP) delivery and truncated gRNAs. This review article provides extensive information to cautiously interpret off-target effects to assist the basic and clinical applications in biomedicine.

Integral membrane proteins (IMPs) fulfill important physiological functions by providing cell–environment, cell–cell and virus–host communication; nutrients intake; export of toxic compounds out of cells; and more. However, some IMPs have obliterated functions due to polypeptide mutations, modifications in membrane properties and/or other environmental factors—resulting in damaged binding to ligands and the adoption of non-physiological conformations that prevent the protein from returning to its physiological state. Thus, elucidating IMPs’ mechanisms of function and malfunction at the molecular level is important for enhancing our understanding of cell and organism physiology. This understanding also helps pharmaceutical developments for restoring or inhibiting protein activity. To this end, in vitro studies provide invaluable information about IMPs’ structure and the relation between structural dynamics and function. Typically, these studies are conducted on transferred from native membranes to membrane-mimicking nano-platforms (membrane mimetics) purified IMPs. Here, we review the most widely used membrane mimetics in structural and functional studies of IMPs. These membrane mimetics are detergents, liposomes, bicelles, nanodiscs/Lipodisqs, amphipols, and lipidic cubic phases. We also discuss the protocols for IMPs reconstitution in membrane mimetics as well as the applicability of these membrane mimetic-IMP complexes in studies via a variety of biochemical, biophysical, and structural biology techniques.

Metabolites and their pathways are central for adaptation and survival. Metabolic modeling elucidates in silico all the possible flux pathways (flux balance analysis, FBA) and predicts the actual fluxes under a given situation, further refinement of these models is possible by including experimental isotopologue data. In this review, we initially introduce the key theoretical concepts and different analysis steps in the modeling process before comparing flux calculation and metabolite analysis programs such as C13, BioOpt, COBRA toolbox, Metatool, efmtool, FiatFlux, ReMatch, VANTED, iMAT and YANA. Their respective strengths and limitations are discussed and compared to alternative software. While data analysis of metabolites, calculation of metabolic fluxes, pathways and their condition-specific changes are all possible, we highlight the considerations that need to be taken into account before deciding on a specific software. Current challenges in the field include the computation of large-scale networks (in elementary mode analysis), regulatory interactions and detailed kinetics, and these are discussed in the light of powerful new approaches.

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The HIV-1 encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with multiple host proteins to support virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. The structures of full-length Vpu in its monomeric and oligomeric forms are unknown, although both the monomer and oligomer are deemed important. Here, we report on the diversity of Vpu oligomeric structures and how the environment affects the Vpu oligomer formation. We produced a uniquely designed MBP-Vpu chimera protein in E. coli in soluble form. We subjected this protein to analytical size exclusion chromatography (SEC) and negative staining electron microscopy (nsEM). Strikingly, we found that MBP-Vpu forms stable oligomers in solution, presumably driven by Vpu transmembrane domain self-association. Our coarse modeling suggests that these oligomers are pentamers, in agreement with the pentameric membrane-bound Vpu. To the best of our knowledge, this is the first observation of Vpu self-association out of lipid membrane environment. We further found that MBP-Vpu oligomer stability decreases when the protein was reconstituted in lipid membrane mimetics, such as β-DDM, and mixtures of lyso PC/PG or DHPC/DHPG. In these cases, significant oligomer heterogeneity was observed with oligomeric order lesser than that of MBP-Vpu oligomer in solution, but larger oligomers were observed as well. Importantly, we found that in lyso PC/PG, above certain protein concentration, MBP-Vpu forms linear array-like structures, which is also novel. Thus, our studies provide unique information about Vpu protein quaternary organization by capturing multiple Vpu oligomeric structures, which we believe are physiologically relevant.

post-translocation configuration within the ribosomal peptidyl-tRNA binding site that is distinct from proL. Collectively, our results demonstrate that sufB2 promotes þ1 frameshifting during EF-G-dependent translocation, thus providing unique insights into the timing, location, and mechanism of þ1 frameshifting and revealing ways in which the ribosome might be engineered to increase the efficiency of UAA incorporation.

We investigate the molecular mechanisms of the Mycobacterium tuberculosis (Mtb) EfpA membrane exporter. EfpA is a ca. 56 kDa monomeric integral membrane protein, which belongs to the Major Facilitator Superfamily transporters, and the QacA subfamily. It has fourteen predicted transmembrane segments/helices (TMs) with both N‐and C‐termini located in the intracellular space. EfpA exports anti‐Mtb drugs (e.g., isoniazid, fluoroquinolones, rifampicin, tetracycline, clofazimine) out of the cell coupled to the antiport of H+. Thus, EfpA supports a major mechanism of Mtb drug resistance. However, its structure and structure‐function relationship are currently poorly understood. What is more, to the best of our knowledge, the protein was not produced recombinantly in quantities needed for large‐scale in vitro investigations. To overcome this deficiency, we cloned the gene encoding EfpA and expressed the protein in E. coli host. Two fusion constructs of EfpA with respectively His8‐FLAG tag and His8‐maltose binding protein (MBP) tags were produced. These tags were designed to facilitate protein purification utilizing double affinity chromatography. We used SDS‐PAGE and western blotting to identify the protein localization in E. coli cellular fractions. We found that although these fusion constructs were expressed to a minor extent in the E. coli plasma membrane and can be partially purified, the proteins were mostly deposited in inclusion bodies, similarly to what was observed previously for other Mtb membrane proteins. We purified to a high degree and refolded these proteins from inclusion bodies. Currently, we are working on producing large quantities of highly‐pure EfpA protein for functional and structural studies. We further used Swiss Model program and the structures of the distant homologues NorC transporter and peptide transporter DtpA solved in outward‐ and inward‐facing states, respectively, to predict the structure of EfpA in the two major functionally‐relevant conformations. After that, we designed two double cysteine EfpA mutants E141C/F398C and F170C/Q401C, which report on large conformational rearrangements upon transition from outward‐ to inward‐facing conformations, for spin labeling and electron paramagnetic resonance (EPR) study. These mutants were generated via site‐directed mutagenesis on the background of cysteine‐free EfpA construct and are being produced in E. coli. We will further discuss results from EfpA‐drug binding and EPR experiments. Our studies have the potential to significantly advance the detailed characterization of EfpA functional mechanism.

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