GeneLancet Biosciences is developing LgRNA conjugate-guided precise genome editors, including DuoSTAR Editors (segNA-directed polymerase editors) as one-time-cures for neurodegenerative disorders, KRAS-driven cancers, Hepatocellular Carcinoma (HCC) and chronic viral infections.

STAR (Seek-Tag-Amend-Release) is a precise multiple-turnover genome editor for editing target genes with enhanced release of the acting ribonucleoprotein (RNP) complex. 1. Seek: a nCas9(H840A)/segNA complex binds target DNA that contains a sequence match to the first 17-20 nucleotides of the segNA and immediately before a protospacer adjacent motif (PAM) to form an R-loop. 2. Tag: cleavage and further 3’-end processing by RuvC leave a PAM distal strand of various lengths which is asymmetrically released from the nCas9:segNA:DNA complex. The released DNA strand acts as a primer and hybridizes with the 3’- homology arm of the conjugated ssDNA strand, which acts as a template for DNA repair. 3. Amend: the tagged DNA nick is repaired by ssDNA-templated synthesis and the 5’- flaps are removed and the gaps are ligated by cellular enzymes. The editing efficiency can be enhanced by a second nick at non-editing strand, the mismatches in non-editing strand can be efficiently corrected via edited-strand templated DNA synthesis, while indels can be minimal because of the high processivity of DNA polymerase. 4. Release: Cas9:segNA complex is released from the repaired DNA and ready for next cycle.

EpiSTAR is a precise epigenetic editor of single CpG resolution when segNA of methylated ssDNA is used in combination with a deactivated Cas protein.

DuoSTAR (with a segNA pair) enables diverse editing including efficient precise single base editing, deletion of nucleotides and long repeats and insertions of longer DNAs (see the figure).

DuoSTAR is a dual segNA- or (segNA+LgRNA)-guided CRISPR-polymerase editor with a distinct MOA and superior performance:

  1. Multiple turnover editing due to a covalently linked stable ssDNA template
  2. MOA-assured low off-target
  3. No double-strand breaks required
  4. Fusion protein of large sizes is not required (at the presence of cellular enzymes)
  5. Active nuclear transport of transgene ssDNA by STAR ribonucleoprotein (RNP) complex for both episomal expression and as an editing template (applications include two-wave in vivo CAR-X and gene therapy)
  6. Multiplex precise editing for treatments of polygenic disorders