Through rational engineering of the ISCro4 bridge RNA and deep mutational scanning of its recombinase, we achieved up to 20% insertion efficiency into the human genome and genome-wide specificity as high as 82%. We further demonstrated intrachromosomal inversion and excision, mobilizing up to 0.93 megabases of DNA.
Lastly, we provided proof-of-concept for plasmid-based excision of disease-relevant gene regulatory regions or repeat expansions.
As a proof-of-concept, the researchers created artificial DNA constructs containing the same toxic repeat sequences that cause progressive neuromuscular decline in Friedreich's ataxia patients.
While healthy individuals carry fewer than 10 sequential copies of a three-letter DNA sequence, people with the disorder can harbor up to 1,700 copies, which interferes with normal gene function.
The engineered ISCro4 successfully removed these repeats from the artificial constructs, in some cases eliminating over 80% of the expanded sequences.
