Chinese, U.S. Researchers Use Gene Editing to Precisely Insert Large DNA Fragments Without Scars

Researchers in China and the United States have developed a gene-editing technique that precisely inserts large DNA fragments of up to 12,500 base pairs without leaving scars.

According to the international journal Nature Biotechnology on Aug. 28 (local time), a joint research team led by Wang Lingbo of Fudan University and Feng Zhang of the Broad Institute in the United States reported a technique called donor complementary prime editing (DoPE).

DoPE combines a double-stranded DNA donor with a 3′ overhang, a pair of complementary prime editing guide RNAs and the PE2* prime editor to precisely insert DNA sequences of up to 12.5 kilobases (kb).

Existing gene-insertion techniques cause double-strand breaks (DSBs), which can trigger chromosomal rearrangements or DNA damage responses, or require multiple editing steps, resulting in low efficiency.

The researchers said the method can precisely insert large DNA fragments in a single step without recombinases or transposons.

Using a donor pool made from synthetic single-stranded oligonucleotides, the team demonstrated saturation mutagenesis at the amino acid and nucleotide levels at the target EGFP site.

The researchers also presented a method for uniformly correcting different alleles by replacing a mutated exon in the PRKCSH gene.

The technique supports insertions ranging from short fragments to large ones exceeding 10 kb. It can also insert multiple DNA sequences in varied combinations at a single target site, making it potentially useful for gene screening.

The researchers said DoPE could be an alternative to existing PASSIGE, eePASSIGE and PASTE techniques in a range of applications requiring precise insertion of gene-sized DNA fragments.