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Highlights d BioID identifies novel ADAR interactors, including regulators of A-to-I RNA editing d Interactors include all DZF-domain-containing proteins: ILF2, ILF3, STRBP, and ZFR d DZF-domain-containing proteins bind ADAR in an RNAdependent manner d ILF3 is a broadly influential negative regulator of editing

Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity.

Highlights d Drosophila Zn72D is a broadly influential regulator of neuronal A-to-I RNA editing d Zn72D regulates ADAR protein levels and editing levels at hundreds of editing sites d Loss of Zn72D causes morphological defects at the neuromuscular junction d Zn72D regulation of editing is conserved in mammalian neurons