Targeted approaches for gene editing typically require double-strand DNA breaks (DSBs) that can generate unwanted byproducts or lead to chromosomal abnormalities. A further drawback of these methods is that they are often unsuitable for manipulating larger DNA sequences, limiting their utility for therapeutic applications. Twin prime editing (twinPE) is a novel, DSB-independent form of gene editing that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for programmable deletion, substitution, insertion, or inversion of larger DNA sequences within the human genome. Data published in Nature Biotechnology show how twinPE was used to manipulate the genomes of human cells and demonstrate the potential of the technology for studying and treating genetic diseases.
The twinPE strategy
TwinPE increases the efficiency of prime editing by using two pegRNAs, each targeting a different DNA strand, to produce the desired modification. By templating complementary 3’ flaps able to hybridize with one another, the paired pegRNAs enable the production of an intermediate containing annealed 3′ overhangs of the new DNA sequence and annealed 5′ overhangs of the original DNA. Excision of the latter, followed by ligation of the nicks, results in the replacement of the endogenous sequence with the paired 3′ flap sequences. By modifying the template design, the method can be adapted to delete, insert, or invert a specific DNA sequence.
twinPE provides highly efficient conversion
To validate the twinPE strategy, Anzalone et al. targeted the site 3 locus in HEK293T cells (HEK3) to replace a 90-bp endogenous sequence with a 38-bp Bxb1 integrase attB substrate sequence. TriLink’s CleanCap® AG was used for co-transcriptional capping of the pegRNAs, which were designed to generate 3′ flaps with overlapping complementarity ranging from 22-bp to 38-bp. Transfection of pairs of pegRNAs with the reverse transcriptase, PE2, produced highly efficient attB site insertion, with several pegRNA pairs yielding >80% conversion.
Utility for large sequence manipulations
Anzalone et al. next used twinPE for a further series of manipulations. Inserting a 108-bp FKBP12 cDNA fragment into the HEK3 locus achieved a 20-fold greater insertion efficiency over PE3 but represented the upper limit of sequence insertion. To integrate larger DNA sequences, twinPE was combined with a site-specific integrase, allowing insertion of gene-sized DNA plasmids (>5,000 bp) into safe-harbor loci. A recombinase inversion approach enabled targeted sequence inversions of 40 kb, suggesting that twinPE might be combined with site-specific serine recombinases to install or correct large or complex pathogenic gene variants.
Potential for therapeutic targeting
To demonstrate the potential of twinPE for therapeutic targeting, Anzalone et al. performed several experiments to manipulate disease-associated genes in human cells. These included recoding portions of the PAH gene known to harbor mutations in patients with the genetic metabolic disorder phenylketonuria, which achieved efficiencies of 23% for a 46-bp recoding and 27% for a 64-bp recoding, and deletion of a 780-bp sequence containing DMD exon 51, which has been linked to Duchenne muscular dystrophy.
Featured product:: CleanCap® AG
Article Reference:Anzalone AV, Gao XD, Podracky CJ, et al., Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing, https://www.nature.com/articles/s41587-021-01133-w.