Background

Techniques that precisely insert long DNA sequences at specific genomic locations are essential tools in gene therapy and functional genomics. Conventional CRISPR–Cas9-based homology-directed repair (HDR) relies on double-strand breaks (DSBs) in the target DNA. This process can lead to large deletions, insertions/deletions (indels), chromosomal rearrangements, and p53-mediated DNA damage responses. HDR efficiency is also low in slowly dividing cells.

Prime editing uses a Cas9 nickase fused with reverse transcriptase to edit bases without DSBs, but it has limitations in inserting long sequences. Twin prime editing or methods combining recombinase and transposon enzymes have increased insertion capacity but require multi-step editing or additional enzymes and recognition sequences. For saturation mutagenesis analyses that investigate multiple variants simultaneously, complex guide designs were required for each mutation. To overcome these limitations, a research team from Fudan University developed donor-complementary prime editing (DoPE). Nature Biotechnology paper

Key Findings

DoPE consists of a double-stranded DNA donor (odsDNA) with short 3′ single-strand overhangs at both ends, two prime editing guide RNAs (opegRNAs) complementary to the overhangs, and a nuclear localization signal-enhanced PE2* editor. The PE2* creates 3′ flaps on both sides of the target genome that are complementary to the donor ends, and a complementary sequence of approximately 30 nucleotides connects the donor to the target site. Recombinase or transposon enzymes are not used.

The research team replaced a 108-base pair region in the ACTB-EGFP reporter in HEK293T cells to generate a fluorescent protein variant and confirmed insertions in H9 human embryonic stem cells and HeLa cells as well. By combining a single pair of opegRNAs with a synthetic single-stranded oligonucleotide pool, they achieved amino acid- and nucleotide-level saturation mutagenesis analysis in the EGFP target region. Unlike existing prime editing screening methods, this approach allows for changing only the donor library without redesigning the guide RNA for each mutation.

Insertion sizes expanded from dozens of base pairs to 1.4kb, 2.1kb, 3.1kb, and up to 12.5kb. The 1.4kb CMV-EGFP cassette was validated at the AAVS1 and TP53 loci in HEK293T cells and at the AAVS1 locus in H9 cells. The 12.5kb insertion was also confirmed at the AAVS1 and TP53 targets. The odsDNA could be synthesized by combining partially complementary single-stranded DNA or by treating PCR products with protected ends using lambda exonuclease.

Implications and Outlook

In disease models, the mutant exon of PRKCSH associated with autosomal dominant polycystic liver disease was completely replaced with a normal sequence. The research team performed individual or simultaneous exon replacements in HEK293T and H9 cells with different mutations and observed recovery of PRKCSH protein expression after editing. This is a 'mutation-independent' strategy, replacing the entire exon containing the mutation with a normal version, rather than designing correction tools for each mutation.

However, this achievement remains limited to cultured cell experiments. Challenges remain in delivering the long odsDNA and large PE2* together into tissues, in efficiency variations depending on cell type and genomic location, and in evaluating incomplete insertions and off-target integration of the donor. Off-target analyses were limited to computationally predicted candidate sites and cannot replace whole-genome-level safety assessments. The potential conflict of interest, as the research team is preparing a patent for DoPE, should also be considered in comparative studies and reproducibility evaluations.