Transient delivery of CRISPR-based genome editing effectors is important to reduce off-target effects and immune responses. Recently extracellular vesicles (EVs) have been explored for Cas9 ribonucleoprotein (RNP) delivery. However, lack of mechanisms to enrich RNPs into EVs limited the efficiency of EVs as a RNP delivery vehicle. Here we describe a mechanism to actively enrich RNPs into EVs. We used the specific interaction between RNA aptamer and aptamer-binding protein (ABP) to enrich RNPs into EVs. We inserted RNA aptamer com into single guide RNA (sgRNA), and fused com-binding ABP Com to both termini of tetraspan protein CD63 that is abundant in exosomes. We found that the Com/com interaction enriched Cas9 and adenine base editor (ABE) RNPs into EVs, via forming a three-component complex including CD63-Com fusion protein, com-modified sgRNA and Cas9 or ABE. The RNP enriched EVs are efficient in genome editing and transiently expressed. The system is capable of delivering RNPs targeting multiple loci for multiplex genome editing. In addition, Cas9 from different species can be used together. The EV-delivered RNPs are active in vivo. The data show that the aptamer and ABP interactions can be utilized to actively enrich RNPs into EVs for improved genome editing efficiency and safety. K E Y WO R D S adenine base editor, aptamer, aptamer-binding protein, CD63, CRISPR/Cas9, delivery, extracellular vesicle, ribonucleoprotein  INTRODUCTION Extracellular vesicles (EVs) can be broadly divided into two main categories, exosomes and microvesicles, based on the mechanisms of generation. Exosomes are heterogeneous membranous vesicles released by various cells via inward budding of multivesicular bodies and subsequent fusion of the multivesicular body membranes with the plasma membrane (Heijnen et al., 1999). Exosomes play important roles in intercellular crosstalk and disease pathogenesis, and are believed to function by transporting RNAs, proteins and lipids from one cell to the other (Ratajczak et al., 2006). Microvesicles are generated by the outward budding and fission of the plasma membrane and the subsequent release of vesicles into the extracellular space. Exosomes and microvesicles overlap in sizes and currently it is difficult to separate the two types of vesicles in preparation. The transportation capability of EVs prompted the exploration of using EVs as drug delivery vehicles (Alvarez-Erviti et al.

Studies have highlighted the role of nutritional and metabolic modulators in asthma pathobiology. Steroid resistance is an important clinical problem in asthma but lacks good experimental models. Linoleic acid, a polyunsaturated fatty acid, has been linked to asthma and glucocorticoid sensitivity. Its 12/15–lipoxygenase metabolite, 13-S-hydroxyoctadecadienoic acid (HODE) induces mitochondrial dysfunction, with severe airway obstruction and neutrophilic airway inflammation. Here we show that HODE administration leads to steroid unresponsiveness in an otherwise steroid responsive model of allergic airway inflammation (AAI). HODE treatment to allergic mice further increased airway hyperresponsiveness and goblet metaplasia. Treatment with dexamethasone was associated with increased neutrophilic inflammation in HODE treated allergic mice; unlike control allergic mice that showed resolution of inflammation. HODE induced loss of steroid sensitivity was associated with increased p-NFkB in mice and reduced GR-α transcript levels in cultured human bronchial epithelia. In summary, HODE modifies typical AAI to recapitulate many of the phenotypic features seen in severe steroid unresponsive asthma. We speculate that since HODE is a natural metabolite, it may be relevant to the increased asthma severity and steroid insensitivity in patients who are obese or consume high fat diets. Further characterization of HODE induced steroid insensitivity may clarify the mechanisms.

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