Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.

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Municipal wastewater management is an important target area for reducing the spread of antibiotic resistance, especially given the parallel increasing need for water reuse. Anaerobic membrane bioreactors (AnMBRs) have the potential to play a key role in safely expanding non-potable wastewater reuse practices. In the present study, the effect of the commencing treatment of municipal wastewater by an AnMBR was evaluated after an extended startup phase using only synthetic wastewater. Antibiotic resistance genes (ARGs) associated with sulfonamides, tetracyclines, and β-lactams were quantified, and effluent microbial community progression was analyzed. Results indicated that the AnMBR effluent inherently harbored all targeted ARGs prior to the introduction of real wastewater (104–109 copies/100 mL effluent). sul1, sul2, and intI1 genes were notably higher initially than other genes and markedly increased after the system was transitioned to municipal wastewater. Although potentially pathogenic bacteria made up over 20% relative abundance of the influent, AnMBR effluents showed a marginalization of these groups as their microbial communities more closely resembled the tightly bound layer of membrane biofilms. This work highlights the need for emerging treatment systems to be evaluated on a basis that incorporates the differentiation of system-associated ARGs and assesses their environmental transmission potential within the effluent communities.

Treated wastewater has been addressed as an alternative water resource to a great extent lately because of major water scarcity faced worldwide. Among wastewater treatment technologies envisaged, anaerobic membrane bioreactors...