A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular anti-sense oligonucleotide activity

Anti-sense oligonucleotides (ASOs) are modified synthetic single-stranded molecules with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can reduce or alter mRNA expression upon binding of splice site positions. To target RNA in the nucleus or cytoplasm, ASOs must cross membranes, a poorly understood process. We performed an unbiased CRISPR/Cas9 knockout screen with a genetic splice reporter to identify genes that can increase or decrease ASO activity, resulting in the most comprehensive catalog of ASO-activity modifier genes. Here we reveal distinct targets, including AP1M1 and TBC1D23, linking ASO activity to transport of cargo between the Golgi and endosomes. AP1M1 absence strongly increases ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASO residence time in the endosomal system may increase the likelihood of ASO escape. This insight into AP1M1 role in ASO trafficking suggests a way for enhancing the therapeutic efficacy of ASOs by manipulating the endolysosomal pathways.