Some RNA species, especially microRNAs, are non-randomly sorted into exosomes, but how selectivity of RNA exosomal sorting is achieved is unknown. We found that all three variants of RNA-binding ubiquitin E3 ligase (MEX3C)–MEX3C-1, MEX3C-2, and MEX3C-3 –interact with adaptor-related protein complex 2 (AP-2), a cargo adaptor in clathrin-mediated endocytosis. MEX3C’s C-terminal RING finger domain and the hnRNP K homology (KH) domain shared by the three MEX3C variants are both necessary for MEX3C/AP-2 interaction. MEX3C associates with the endolysosomal compartment through an endocytosis-like process. siRNA-mediated inhibition of the MEX3C or AP-2 complex substantially decreased exosomal but not cellular microRNA miR-451a expression. Exosomal sorting is ceramide-dependent but not ESCRT-dependent in microRNA miR-451a. That RNA-binding protein associates with membrane trafficking machinery, and that its involvement in exosomal microRNA expression, suggest the existence of a mechanism for specific recruiting of RNA molecules to endosomes for subsequent exosomal sorting.

The discovery of designer nucleases has made genome editing much more efficient than before. The designer nucleases have been widely used for mechanistic studies, animal model generation and gene therapy development. However, potential off-targets and host immune responses are issues still need to be addressed for in vivo uses, especially clinical applications. Short term expression of the designer nucleases is necessary to reduce both risks. Currently, various delivery methods are being developed for transient expression of designer nucleases including Zinc Finger Nuclease (ZNF), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas). Recently, virus-like particles are being used for gene editing. In this review, we will talk through commonly used genome editing nucleases, discuss gene editing delivery tools and review the latest literature using virus-like particles to deliver gene editing effectors.

The argonaute protein from the thermophilic bacterium Thermus thermophilus shows DNA-guided DNA interfering activity at high temperatures, complicating its application in mammalian cells. A recent work reported that the argonaute protein from Natronobacterium gregoryi (NgAgo) had DNA-guided genome editing activity in mammalian cells. We compared the genome editing activities of NgAgo and Staphylococcus aureus Cas9 (SaCas9) in human HEK293T cells side by side. EGFP reporter assays and DNA sequencing consistently revealed high genome editing activity from SaCas9. However, these assays did not demonstrate genome editing activity by NgAgo. We confirmed that the conditions allowed simultaneous transfection of the NgAgo expressing plasmid DNA and DNA guides, as well as heterologous expression of NgAgo in the HEK293T cells. Our data show that NgAgo is not a robust genome editing tool, although it may have such activity under other conditions.