Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing

Duško Lainšček, Vida Forstnerič, Veronika Mikolič, Špela Malenšek, Peter Pečan, Mojca Benčina, Matjaž Sever, Helena Podgornik and Roman Jerala ()
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Duško Lainšček: National Institute of Chemistry
Vida Forstnerič: National Institute of Chemistry
Veronika Mikolič: University Medical Centre Ljubljana
Špela Malenšek: National Institute of Chemistry
Peter Pečan: National Institute of Chemistry
Mojca Benčina: National Institute of Chemistry
Matjaž Sever: University Medical Centre Ljubljana
Helena Podgornik: University Medical Centre Ljubljana
Roman Jerala: National Institute of Chemistry

Nature Communications, 2022, vol. 13, issue 1, 1-12

Abstract: Abstract The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology.

Date: 2022
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DOI: 10.1038/s41467-022-31386-1

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