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.

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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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