Structural mechanism of programmable gene integration by the R2 retrotransposon coupled with CRISPR-Cas9

R2 retroelements are non-long-terminal-repeat retrotransposons that primarily insert into 28S ribosomal DNA loci across diverse eukaryotes. R2 elements home to their target sites through target-primed reverse transcription (TPRT), in which the R2 protein recognizes and nicks the target DNA to prime reverse transcription using an R2-encoding RNA as the template. The avian R2 system from Taeniopygia guttata (R2Tg) can insert a payload sequence within an RNA template into 28S ribosomal DNA loci in human cells. Here, we present cryo-electron microscopy structures of the R2Tg-template RNA-target DNA complex in the TPRT initiation state, both alone and bound to a Cas9-guide RNA complex. These structures provide mechanistic insights into R2Tg-mediated TPRT in its natural context and in the programmable gene integration system STITCHR (site-specific target-primed insertion through targeted CRISPR homing of retroelements). Furthermore, we combine these structural insights with computational protein design methods to engineer an enhanced STITCHR system.