Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci

Abstract

Current approaches for inserting autonomous transgenes into the genome, such as CRISPR–Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.

Conserved and divergent DNA recognition specificities and functions of R2 retrotransposon N-terminal domains

“…Product DNAs were produced corresponding to first-strand nicking followed by reverse transcription of one or two consecutive 3′ UTR RNA molecules ( Figure 5D , 1X and 2X), resulting from initial TPRT and subsequent elongation of the initial TPRT product by template jumping. 21 , 30 Unexpectedly, both TrCasB and ZoAl proteins lacking the A-clade-specific ZF3 (ΔZF3) or ZF3 and ZF2 (ΔZF3-2) supported robust target-site nicking and TPRT, but no activity was detected for the ΔZF3-1 proteins lacking all N-terminal ZFs ( Figures 5D and 5E ). We conclude that A-clade R2 protein ZF1 and Myb domains are required and sufficient to support accurate first-strand nicking, cDNA synthesis initiation, and processive cDNA synthesis in vitro .…”

“…Avian R2 proteins from the A clade have promising applications in transgene supplementation of the human genome, providing a mechanism for gene insertion into rDNA as a safe harbor. 21 Safe-harbor transgene delivery would complement CRISPR-Cas approaches for endogenous gene editing. A current limitation is that A-clade R2 protein domains and rDNA sequence elements that support high insertion-site specificity have not been explored.…”

CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery

“…In addition, insertion at rDNA site may result in reduced expression over time due to rDNA array instability 36 . An attempt to integrate R2 elements with Cas9 for RNA-guided reverse transcription and integration was unsuccessful to achieve complete cDNA synthesis and integration 25 .…”

“…However, R2 elements are naturally restricted to the ribosomal DNA (rDNA) loci, which limits their utility for editing in other genomic locations. In addition, insertion at rDNA site may result in reduced expression over time due to rDNA array instability 36 . An attempt to integrate R2 elements with Cas9 for RNA-guided reverse transcription and integration was unsuccessful to achieve complete cDNA synthesis and integration 25 .…”

Allele Sails: launching traits and fates into wild populations with DNA sequence modifiers

“…As another point of reference, as of 2019, half of the pathogenic variants in the human ClinVar database were point-mutations, and almost 90% of clinically relevant insertions and deletions were less than 30bp 60 . Finally, recent work shows that larger fragments of DNA (which would albeit be considered transgenes) can be copy-pasted from one location to another using engineered retrotransposons 61,62 . These observations suggest it may soon be possible to push larger fragments of DNA into a population in a self-limiting manner using an editing locus transmitted in a Mendelian manner…”