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.

Alzheimer's disease (AD) chiefly characterizes a progressively neurodegenerative disorder of the brain, and eventually leads to irreversible loss of intellectual abilities. The β-amyloid (Aβ)-induced neurodegeneration is believed to be the main pathological mechanism of AD, and Aβ production inhibition or its clearance promotion is one of the promising therapeutic strategies for anti-AD research. Here, we report that the natural product arctigenin from Arctium lappa (L.) can both inhibit Aβ production by suppressing β-site amyloid precursor protein cleavage enzyme 1 expression and promote Aβ clearance by enhancing autophagy through AKT/mTOR signaling inhibition and AMPK/Raptor pathway activation as investigated in cells and APP/PS1 transgenic AD model mice. Moreover, the results showing that treatment of arctigenin in mice highly decreased Aβ formation and senile plaques and efficiently ameliorated AD mouse memory impairment strongly highlight the potential of arctigenin in anti-AD drug discovery.