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This patent describes append editing, a genome-editing method that combines a programmable Cas protein with the DNA ADP-ribosyltransferase DarT to install ADP-ribose at a selected thymine. The targeted modification recruits endogenous DNA-repair pathways, enabling homologous recombination in bacteria and direct T-to-A or T-to-C substitutions in eukaryotic cells. By avoiding double-strand DNA breaks, append editing offers a potentially safer and more versatile alternative to conventional nuclease-based editing while expanding the range of accessible base conversions.
This patent describes a universal riboswitch for inducible gene expression based on a ligand-responsive, self-splicing T4 td intron, called SIBR. The engineered intron can be inserted into different positions within a gene and, upon addition of an inducer such as theophylline, splices itself from the RNA to restore expression of the encoded protein. The system can regulate proteins, RNAs and CRISPR–Cas genome-editing components across diverse prokaryotic and eukaryotic hosts. Unlike conventional riboswitches placed in the 5′ untranslated region, it is independent of the Shine–Dalgarno sequence and can be incorporated without changing the final protein sequence, making it particularly valuable for non-model organisms that lack reliable inducible promoters.