Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Current gene editing methods often rely on double-strand DNA breaks (DSBs), which can cause unintended genetic alterations like indels and chromosomal abnormalities.
• These limitations hinder the precise study and therapeutic application of genomic sequence manipulation for human genetic diseases.

Purpose of the Study:

• To introduce twin prime editing (twinPE), a novel gene editing technology that circumvents the need for DSBs.
• To demonstrate twinPE's capability for precise DNA sequence manipulation, including replacement, excision, integration, and inversion at endogenous genomic sites.

Main Methods:

• Developed twin prime editing (twinPE), a method utilizing a prime editor protein and two prime editing guide RNAs (pegRNAs).
• The two pegRNAs guide the synthesis of complementary DNA flaps that replace targeted endogenous sequences between nick sites.
• Combined twinPE with a site-specific serine recombinase for integrating large DNA fragments and performing large-scale sequence inversions.

Main Results:

• TwinPE successfully achieved programmable replacement and excision of DNA sequences at endogenous human genomic sites without inducing DSBs.
• The integration of gene-sized DNA plasmids (>5,000 bp) and targeted inversion of a 40 kb sequence were accomplished in human cells.
• Demonstrated the versatility and precision of twinPE for complex genomic modifications.

Conclusions:

• TwinPE represents a significant advancement in precision gene editing, offering a DSB-independent alternative.
• This technology expands the toolkit for correcting or complementing large and complex pathogenic alleles, potentially improving therapeutic strategies for genetic disorders.