Template-jumping prime editing enables large insertion and exon rewriting in vivo

Abstract

Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50 bp) sequences. Employing paired prime editing guide RNAs (pegRNAs) has enabled PE to better mediate relatively large insertions in vitro, but the efficiency of larger insertions (>400 bp) remains low and in vivo application has not been demonstrated. Inspired by the efficient genomic insertion mechanism of retrotransposons, we develop a template-jumping (TJ) PE approach for the insertion of large DNA fragments using a single pegRNA. TJ-pegRNA harbors the insertion sequence as well as two primer binding sites (PBSs), with one PBS matching a nicking sgRNA site. TJ-PE precisely inserts 200 bp and 500 bp fragments with up to 50.5 and 11.4% efficiency, respectively, and enables GFP (~800 bp) insertion and expression in cells. We transcribe split circular TJ-petRNA in vitro via a permuted group I catalytic intron for non-viral delivery in cells. Finally, we demonstrate that TJ-PE can rewrite an exon in the liver of tyrosinemia I mice to reverse the disease phenotype. TJ-PE has the potential to insert large DNA fragments without double-stranded DNA breaks and facilitate mutation hotspot exon rewriting in vivo.

Fig. 1. Retrotransposon-like template jump prime editing (TJ-PE) mediates large genomic insertions.

a Schematics of non-LTR (left) and TJ-PE (right) mechanisms. TJ-pegRNA: template jump prime editing guide RNA; PBS1: primer binding site 1; RC-PBS2: reverse complement sequence of PBS2. b Insertion of DNA fragments with PE3 control or TJ-PE at AAVS1 site. HEK293T cells were transfected with PE2, nicking sgRNA, and either TJ-pegRNA (TJ-PE) or control pegRNA (PE3). PCR using primers flanking AAVS1 detected amplicons of 200, 300, and 500-bp insertions with a deletion of 90 bp at the AAVS1 locus. Insertion bands of expected size are denoted with arrows. Ins: insertion, WT: wild-type. c Insertion efficiency at AAVS1 locus measured by ddPCR. Results were obtained from three independent experiments, shown as mean ± s.d. d The insertion bands were gel purified. Sanger sequencing shows accurate insertions. e Precise insertion was confirmed by TA cloning and Sanger sequencing of 12 individual clones. f Absolute total precise insertion efficiency of a 200-bp DNA fragment at the AAVS1 locus was quantified by deep sequencing. Results were obtained from three independent experiments (n = 3), shown as mean ± s.d.

Fig. 2. TJ-PE mediates insertions at multiple genomic loci.

a Insertion of a 200-bp DNA fragment at HEK3 locus by TJ-PE. HEK293T cells were transfected with PE2, nicking sgRNA, and either pegRNA with a control RC-PBS2 (ctrl-RC-PBS2) or a control nicking sgRNA (ctrl-NK) as controls. The insertion band of the predicted size was observed following TJ-PE treatment but not controls (arrow). b Insertion efficiency at HEK3 measured by ddPCR. c Insertion of DNA fragments with PE3 control (pegRNA with a control RC-PBS2 sequence) or TJ-PE at PRNP (left) and IDS (right) loci. Insertion efficiency was measured by ddPCR. Results were obtained from three independent experiments (n = 3), shown as mean ± s.d.

Fig. 3. TJ-PE mediates GFP reporter and functional gene insertion.

a A diagram of the TLR-MCV1 reporter line. Inserting an 89-bp sequence to replace the 39-bp non-functional sequence results in GFP expression. Indels result in mCherry expression. Del: deletion. b PE3 control and TJ-PE were tested in the TLR-MCV1 reporter line, and flow cytometry was used to determine the percentage of fluorescent cells. Results were obtained from three independent experiments, shown as mean ± s.d. c Schematics of TJ-pegRNA and targeting strategy for inserting SA-GOI at AAVS1 locus. SA: splice acceptor; GOI: gene of interest. d Bright field and fluorescence images of HEK293T cells 4 days after transfection with PE, TJ-pegRNA, and nicking sgRNA. HEK293T cells transfected with PE plasmid only served as a control (ctrl). Experiments were done two times, and one is shown. Scale bar: 100 µm. e Efficiency of SA-GFP insertion measured by flow cytometry. Results obtained from three independent experiments, shown as mean ± s.d. f Agarose gel of PCR amplicons showing SA-GFP and SA-Puro insertion. Puro: puromycin. The insertion bands of expected sizes are indicated with arrow. The nonspecific bands are indicated with asterisk. Experiments were done two times, and one is shown.

Fig. 4. In vitro transcribed split circular TJ-petRNA enables large insertion.

a Illustration of split circular TJ-petRNA. The prime editing template RNA (petRNA) sequence carrying an RTT-PBS sequence and an MS2 stem-loop aptamer, and circularized via a permuted group I catalytic intron. Yellow: circularization sequence. b Model of split circular petRNA function in PE. c Urea polyacrylamide gel showing split circular TJ-petRNA after splicing, RNase H, and RNase R digestion. Linear, but not circular, RNA is digested by RNase R. Experiments were done two times, and one is shown. d Editing efficiency of split circular TJ-petRNA at the AAVS1 locus. Synthesized sgRNAs and in vitro transcribed split circular petRNA were co-transfected with nCas9 and MCP-RT mRNA in HEK293T cells. For comparison purposes, HEK293T cells were transfected with PE2, TJ-pegRNA, and nicking sgRNA plasmids. FL-pegRNA: in vitro transcribed full-length TJ-pegRNA. Results were obtained from three independent experiments, shown as mean ± s.d.

Fig. 5. TJ-PE rewrites a correction exon in mouse liver.

a Diagram of Fah splicing before and after correction by TJ-PE. b A diagram of the TJ-PE strategy at Fah locus. c TJ-PE treatment rescues body weight after NTBC withdrawal. The body weight ratio is normalized to day 0 of NTBC withdrawal. NC: treated with Saline. d Schematic of the split-intein dual AAV8 system and tail vein injection experimental timeline. Four-week-old tyrosinemia I mice were injected with a total of 2 × 10¹² vg AAV8. e Representative FAH IHC images. Scale bars, 100 μm. Mice treated with saline were used as negative controls. The lower panel of AAV is a high-magnification view (box with black line). f Quantification of FAH⁺ hepatocytes by IHC six weeks after AAV injection. Data represent mean ± SD. (n = 4 mice for each group).