Dr Ralf Kühn, Max Delbrück Center for Molecular Medicine; Eric Danner, Max Delbrück Center for Molecular Medicine
Advances in genome sequencing and targeted editing technologies have enhanced the feasibility of genome engineering approaches, especially with respect to potential future therapeutic applications. Nevertheless, there are still major limitations in terms of editing efficiencies and a broad access to relevant and desired cell types. Current cell manipulation and therapy approaches that aim for a site-specific exchange of DNA sections are based on classical homologous recombination strategies for precise and controlled sequence replacement. As homology directed repair (HDR) is not readily accessible to non-dividing cells, which are composing most of the adult tissue, this creates a barrier for the development of novel treatments for a wide range of genetic disorders as well as for the understanding of fundamental biological principles. Thus, new editing technologies are required that can overcome existing constraints.
The novel genetic replacement strategy HIROS (Homology Independent Replacement of Sequences) is based on CRISPR/Cas9 genome modifications by selecting suitable guide RNAs that excise a desired genomic fragment by creating two double strand breaks in the presence of a donor plasmid. The donor plasmid carries the substitute fragment and is designed to be cut in parallel releasing such fragment. Flanked by the double stranded breaks the presence of the substitute fragment leads to a high rate of sequence replacement (about 30%) through integration into the gap created by excision of the undesired genomic DNA segment. It has been shown that this replacement strategy is achieved by the non-homologous end joining (NHEJ) pathway of DNA repair. Consequently, the novel DNA replacement strategy is not dependent on homology sequences. Nevertheless, it can still be applied in a targeted manner promoting directed sequence replacement in the correct orientation. In contrast to the general belief that NHEJ is an error-prone process it has been demonstrated that in presence of a substitute fragment this replacement is mainly error-free. Thus, this new methodology opens up a whole new field of genomic manipulation and repair strategies, as NHEJ is not associated with particular phases of the cell cycle. In particular, non-dividing cells can be efficiently and easily addressed for the first time creating the potential for new avenues for basic research as well as for therapeutic and cell therapy approaches in the future.
The technology is offered for in-licensing or collaborative development.
Proof of concept has been demonstrated by using fluorescent reporter systems in genetically modified HeLa and HEK293 cells. Cas9 and substitute DNA delivery has been performed by transfection of plasmids and/or minicircles. Replacement efficiencies of up to 30% have been demonstrated resulting in up to 90% of correct replacement events.
A European priority application (EP17209549.9) has been filed in January 2018.
Manuscript in preparation.
Outline of the novel replacement strategy (HIROS) based on DNA repair by NHEJ
The figure shows the excision of a genomic sequence segment [e.g. an exon harbouring a mutation (*)] by two flanking DSBs created by Cas9 and specific guide(g)RNAs. Non-homologous end joining (NHEJ) is available throughout all phases (except M - mitosis) whereas homology directed repair (HDR) by the homologous recombination pathway occurs only in the S and G2 phases in addition to NHEJ. DSB repair through homologous recombination requires a repair vector which includes a wildtype exon sequences identical to the genomic region flanking the DSBs. The novel HIROS strategy relies on the NHEJ DSB repair pathway which is available in dividing and non-dividing cells. Sequence replacement is achieved by an external, e.g., minicircle vector opened by Cas9. The generated fragment including a wildtype intron is ligated by NHEJ enzymes into the genomic gap. Small sequence duplications remain within the intron sequences but should not affect gene function.