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Twin Prime Editing Promises More Precise DNA Changes Without Double-Strand Breaks

NEW YORK – Researchers at the Broad Institute have developed a new prime editing method that allows for more precise replacement or excision of DNA sequences at endogenous human genomic sites, without the need for double-strand DNA breaks, or DSBs.

In a paper published Thursday in Nature Biotechnology, researchers led by David Liu — who developed prime editing in 2019 — described twin prime editing (twinPE), a method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for programmable replacement or excision of endogenous DNA sequences.

Because the method doesn't require DSBs — which can have unintended consequences such as uncontrolled indel mixtures and chromosomal abnormalities — it can be used for more precise targeted deletion, replacement, integration, or inversion of genomic sequences. This could be useful in the study or treatment of human genetic diseases, the researchers said.

Prime editors can precisely make any of the 12 possible base substitutions, as well as introduce small insertions and deletions, without requiring DSBs. They are also capable of making precise insertions of up to about 40 bp and deletions of up to about 80 bp in human cells with high ratios of desired edits to byproducts, the researchers noted. But even though prime editing is flexible enough to replace one DNA sequence with another, second- and third-generation PE systems haven't been able to mediate insertions or deletions the size of typical gene coding sequences. Such large DNA changes may require long pegRNA reverse transcription templates and long-range DNA polymerization, which would cut into the system's efficiency.

Conversely, site-specific recombinases, or SSRs, can excise, invert, and integrate large DNA sequences in mammalian cells, but reprogramming SSRs has long presented a challenge for genome editing researchers, and has consequently limited their use in precision gene editing applications.

TwinPE's two pegRNAs work by creating a template for the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled the targeted integration of gene-sized DNA plasmids and targeted sequence inversions of 40 kilobases in human cells.

"TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles," the authors wrote.

To test the twinPE strategy, the researchers initially targeted the site 3 locus in HEK293T cells (HEK3) to replace 90 bp of endogenous sequence with a 38-bp substrate sequence. When both pegRNAs were transfected into HEK293T cells along with the second-generation PE system, the researchers observed highly efficient insertion of the substrate sequence, with some combinations of pegRNAs yielding more than 80 percent conversion to the desired product.

A similar strategy for the replacement of the 90-bp endogenous sequence with a 50-bp attachment sequence achieved editing efficiencies up to 58 percent, they added.

To test if twinPE could support the insertion of DNA sequences larger than those that previous studies had demonstrated using second- or third-generation PE systems, the researchers then compared the ability of twinPE and third-generation PE to generate a 108-bp cDNA fragment insertion in the HEK3 locus.

They only achieved inefficient insertion (0.8 percent) of the 108-bp fragment with the PE system alone, whereas twinPE enabled 16 percent insertion efficiency with concomitant deletion of 90 bp of HEK3 sequence, a 20-fold improvement.

"These results demonstrate that twinPE can replace stretches of dozens of nucleotides in human cells with a single pair of pegRNAs," the authors wrote. "In addition to insertion and replacement of DNA sequences, twinPE might also mediate precise deletions more effectively and with greater flexibility than previously described methods."

When they examined potential off-target editing by twinPE, the researchers found no detectable off-target edits or indels at any of four off-target sites beyond background levels in untreated control samples.

"In summary, twinPE expands the capabilities of prime editing to include targeted deletion, replacement, and, when combined with site-specific recombinases, gene-sized integration and inversion without requiring double-strand breaks," the authors concluded. "These new capabilities might enable strategies to study and treat genetic diseases arising from loss-of-function or complex structural mutations."