Site-specific Non-LTR Retrotransposon Mechanisms of Mobility and Transgene Insertion - ABSTRACT Animal genomes are replete with sequence added by the cDNA synthesis activity of non-LTR retrotransposon reverse transcriptases (RTs). These enzymes insert sequence directly into the genome, using a nick to initiate cDNA synthesis on an RNA template, a different strategy than the better-studied integration of an extra- genomic, double-stranded DNA intermediate in LTR retrotransposon and retrovirus life cycles. The single catalytic protein encoded by non-LTR retrotransposons evolved coordinated RNA binding, DNA binding, nicking endonuclease, and RT activities. The enzyme of the human non-LTR retrotransposon LINE-1 inserts new DNA with minimal specificity for target site sequence, allowing it to generate a third of our genome and to continue the mobility that imposes human disease. On the other hand, more ancestral non-LTR retrotransposon proteins generally have target-site specificity for safe-harbor loci in their host genome, which is enabling for their evolutionary persistence. Recently we harnessed an avian R2 non-LTR retrotransposon protein (R2p) to direct site-specific insertion of autonomously expressed transgenes at safe-harbor rDNA loci in the human genome. These rDNA loci generate the precursor of 3 ribosomal RNAs, so they are present in hundreds of copies per genome with the consequence that disruption of a few units is not deleterious. Our method, termed PRINT (Precise RNA-mediated Insertion of Transgenes), requires delivery of only two RNAs: an mRNA encoding R2p and a template RNA encoding the transgene flanked by compact 5’ and 3’ RNA modules. We exploited PRINT to determine mechanisms for the “fill-in” second-strand synthesis necessary to complete gene insertion, which had remained a mystery despite importance for general genome repair. We will use biochemistry, biophysical approaches, PRINT, and native R2 insertion assays developed in the lab, as well cryoEM structures and a newly annotated “zoo” of >300 R2s across the breadth of animal phylogeny, to fulfill 2 synergistic, impact-generating, broad goals: (i) filling knowledge gaps about non-LTR retrotransposon protein structure/function and the cellular mechanisms involved in gene insertion, and (ii) advancing PRINT as an approach for therapeutic gene delivery. Safe-harbor transgene insertion using PRINT is an approach to disease therapy that is complementary to gene disruption or nucleotide correction by CRISPR/Cas [or other method of] introduction of a DNA break, base editing, or prime editing. As additional motivations, the proposed work will (iii) elucidate biochemical and structural principles for unusual nuclease and polymerase activities and highly specific protein-nucleic acid interactions, (iv) illuminate new principles of RNP subcellular trafficking, and (v) define DNA repair pathways and transgene expression features that are specific to nucleolar rDNA versus general chromatin as a transgene location. Overall our studies will increase the efficiency, safety, and application range of a promising genome engineering therapy for human loss-of- function diseases, as well as inform mechanisms used by our deleteriously mobile retrotransposon LINE-1.
