High efficiency of target gene inactivation is one of the key applications of CRISPR, such as targeting primary cells for therapy (e.g. cancer therapy but also for some other genetic disorders).

Gene inactivation depends on the endogenous non-homologous repair processes that may repair the Cas9-triggered double stranded DNA cut without indels, which strongly decreases the efficiency.

We present a strategy to couple Cas9 to the exonuclease to promote large deletions at the cancer-specific target genomic site which increases the efficiency of gene inactivation. This results in a potent increase of deletion formation compared to the standard CRISPR/Cas.

H1 Dlainscek



The role of exonucleases

The exonuclease cleaves off nucleotides one at a time from 3’ or 5’ ends of the polynucleotide chain truncating the chain (DNA or RNA).  

To improve the rate of genetic lesion formation, Cas9 and an exonuclease EXOIII were connected via coiled-coil heterodimer forming peptides, bringing the two enzymes into proximity. The efficiency of our patent-pending protected strategy was better than coexpression of Cas9 and nuclease or single chain genetic fusion between Cas9 and exonuclease. 

Cas Cc Exo

What makes this technology advanced

Improved DNA recession and increased efficiency of gene inactivation


Modular tethering of exonucleases with Cas9 and its variants via coiled-coils


Functional with different Cas nucleases


No additional increase in off-target effects, no cytotoxicity


The possibility of delivery as nucleic acids or as RNPs


Intellectual Property

Priority date EP patent application, September 2019

PCT phase, currently

H Ip

The global gene editing market is growing with more than 15% CAGR and what propels it are multiple applications; from drug discovery processes and high-value personalized medicines to microorganisms and plant genetic engineering. Another market driver are increasingly accessible gene editing tools. CRISPR/Cas is a key disruptive technology, holding about 50% of the market due to its multiplexing ability and a relatively easy design.  

NIH, National Human Genome Research Institute

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