An RNA-based system known as PRINT can create and introduce site-specific, safe, and stable transgenes into the human genome. With just two in vitro transcribed RNAs, PRINT—which stands for “precise RNA-mediated insertion of transgenes”—synthesizes transgenes directly into the genome at a multicopy safe-harbor locus using site-specific primed reverse transcription. This RNA-based strategy can reduce harmful immune responses and guard against random genome insertions from extragenomic DNA for uses like gene therapy—all at a comparatively low cost with quick, scalable production.

The research article “Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci” was published in Nature Biotechnology.

RNA-based transgenesis

Some non-long terminal repeat (non-LTR) retroelements, which use an RNA template for new gene synthesis, show exquisite insertion-site specificity of synthesized complementary DNA (cDNA) directly into the genome using target-primed reverse transcription (TPRT) without generating blunt duplex ends prone to mutagenic re-ligation by canonical nonhomologous end joining.

Co-lead authors Xiaozhu Zhang and Briana Van Treeck from the University of California, Berkeley, developed an approach utilizing RNAs encoding an avian R2 retroelement and 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. 

PRINT inserts specifically within the multicopy ribosomal RNA (rRNA) gene locus (rDNA) transcribed by RNA polymerase (RNAP), which has been used for long-term expression of transgenes. Because the target sequence is in a gene present in hundreds of copies per genome, which in human cells are in tandem arrays that constitute the short arms of five acrocentric chromosomes, cell function is not compromised by retroelement-insertion-mediated inactivation of a few, or in some organisms at least half, of the rDNA units.

When using PRINT on a cultured human primary cell line, Zhang and Van Treeck report that over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. Off-target events and cDNA synthesis errors were extremely rare, detecting only one error per roughly 10,000 bp, consistent with the expected fidelity of RNA and cDNA synthesis.

While the results above constitute proof-of-principle for a delivery approach based on two in vitro transcribed RNAs to supplement the human genome with transgenes of choice, critical knowledge gaps and directions for PRINT development remain to be addressed. For example, it will be essential to monitor and minimize undesirable cellular responses to introduced RNA, long-term transgene persistence should be investigated in contexts relevant to disease therapy, and PRINT efficiency across cell types, including nondividing cells, is interesting to investigate and understand. 

The authors suggest that, if PRINT is eventually developed into a gene therapy approach, it will complement rather than replace CRISPR–Cas-based methods of gene disruption, base editing, and sequence replacement that use a guide RNA to bring DNA synthesis and repair machinery to an endogenous gene locus.

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