DNA-bound avian R2 non-LTR retrotransposon protein recruits a second R2 protein for genome-protective second-strand nicking

Animal genomes have expanded with the mobility of non-long-terminal-repeat retrotransposons. These retrotransposons initiate genome insertions using a retrotransposon protein that coordinately nicks a target site and uses the nick to prime first-strand cDNA synthesis with bound template RNA. To generate a stable genome insertion, a second nick is required and second-strand synthesis involving DNA repair machinery. Here, we use step-wise biochemical reconstitution, cellular assays, and quantitative genomics to establish the second-strand nicking mechanism that supports precise human genome insertions by avian R2 non-LTR retrotransposon protein (R2p). We show that R2p bound to its target site recruits another R2p to nick the second strand, with recruitment reliant on availability of protein domains liberated from RNA by cDNA synthesis. We biochemically screened for side-chain substitutions that selectively crippled or eliminated second-strand nicking and then used these variants to investigate the significance of R2p second-strand nicking in cells. Human genome insertions were assayed using codelivery of mRNA encoding R2p and template RNA encoding a transgene [precise RNA-mediated insertion of transgenes (PRINT)]. R2p variants compromised for second-strand nicking had lower transgene insertion efficiency, yet insertions could occur without R2p-mediated second-strand nicking. Transgene junction profiling revealed an unexpected shift in target-site consequences from small deletions to large duplications obliging extensive DNA repair. This insertion imprecision was rescued by coexpressing the R2p selectively deficient for second-strand nicking with a truncated R2p capable only of second-strand nicking. These insights inform mechanisms that underlie retrotransposon mobility and enable PRINT optimization for therapeutic transgene supplementation of the human genome.