Increasing HDR efficiency: Methods to drastically increase homology-directed repair (HDR) efficiency for precise genome editing

Challenge

Precise genome editing (PGE) using CRISPR/Cas and other genetic scissors sparked hopes to cure many devastating disease. Editing of cells relies on selectively introduced DNA double-strand breaks that are repaired by two competing pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR). Importantly, NHEJ is error-prone and is useful for gene inactivation rather than for PGE. Moreover, the cellular rate of NHEJ is inherently more efficient and is therefore limiting the rate of HDR-dependent PGE. In several attempts, researchers tried to increase PGE efficiency either by enhancing HDR or by inhibiting NHEJ. Unfortunately, the success of these strategies was only moderate, thus the isolation of successfully edited cells remains challenging and simultaneous precise editing of more than one gene is not possible so far.

Technology

Scientists at the Max-Planck Institute for Evolutionary Anthropology in Leipzig have developed two approaches to drastically enhance PGE efficiency. Both take advantage of the crucial role of the kinase DNA-PKcs in promoting NHEJ. Approach I relies on small molecule compound mixtures (called iCRISPY mix). Such mixtures contain an inhibitor of DNA-PKcs as major component. The transient application enables HDR efficiencies to up to 90%. To our knowledge this is the highest efficiency of PGE ever reported in animal cells. Approach II constitutes of genetically modified cell lines harboring mutated DNA-PKcs. These cell lines permit high HDR-rates while confidently maintaining genome stability.

1. Small molecule compounds: iCRISPY mix

Potency of the iCRISPY mix was demonstrated in stem cells and blood cells. It constitutes of a cocktail of small molecules with NHEJ-inhibiting or HDR-enhancing function. Recently, we advanced our original CRISPY mix by a novel very potent NHEJ-inhibitor yielding the improved iCRISPY mix. Sole application of the novel inhibitor M reaches already up to 80% HDR for certain targets. This comes with considerably low acute toxicity and no long-term genotoxicity. For general application, addition of the other components of the iCRISPY mix is advisable and may even further increase this rate. Up to 90% HDR were reached when stem cells and Cpf1 or Cas9 nickase were used. Thus, iCRISPY mix significantly increases HDR-rates independently of cell types or CRISPR enzymes used.

2. Genetic trait enabling the generation of cell lines with enhanced HDR-rates

Cell lines with inherently enhanced HDR-rates would suit much better for certain screenings or other experimental settings. Our scientists discovered that a specific DNA-PKcs mutant acts similarly to the inhibitory compound M. Respective genetic modifications can yield up to 80% HDR efficiency. Combination of this genetic modification with the CRISPY mix can maximize HDR rates to up to 90%. Even heavily pursued multiplexed precise genome editing was successfully demonstrated with mutated cell lines. Importantly, the mutant cell lines maintain genome stability even better than non-mutated wildtype progenitors, probably because error-prone NHEJ is reduced.

We have DNA-PKcs mutant cell lines from 409B2 human pluripotent stem cells, HEK293 cells, and K562 cells readily available.

Commercial Opportunity

We are looking for collaboration and licensing partners interested in the iCRISPY mix and/or in developing cell lines optimized for genome editing.

Development Status

validated research tool

Patent Situation

European priority applications have been filed in 2017 and 2018.

Further Reading

DOI: 10.1038/s41467-018-04609-7