A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells

• Published in: Nucleic acids research Vol. 51; no. 9; pp. 4660 - 4673
• Main Authors: Carusillo, Antonio, Haider, Sibtain, Schäfer, Raul, Rhiel, Manuel, Türk, Daniel, Chmielewski, Kay O, Klermund, Julia, Mosti, Laura, Andrieux, Geoffroy, Schäfer, Richard, Cornu, Tatjana I, Cathomen, Toni, Mussolino, Claudio
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 22.05.2023
• Subjects: CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems - genetics; DNA Breaks, Double-Stranded; DNA End-Joining Repair - genetics; DNA Repair - genetics; Gene Editing; Humans; Recombinational DNA Repair; Synthetic Biology and Bioengineering
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkad255

Abstract Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the increase in error-free editing ranges from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. This novel CRISPR/Cas9 platform accepts clinically relevant repair templates, such as oligodeoxynucleotides (ODNs) and adeno-associated virus (AAV)-based vectors, and has a lower propensity to induce chromosomal translocations as compared to benchmark CRISPR/Cas9. The observed reduced mutational burden, resulting from diminished indel formation at on- and off-target sites, provides a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing. Graphical Abstract Graphical Abstract Upon the generation of a DNA double strand break (DSB) operated by the Cas9 protein, the dominant negative variant of RNF168 (dnRNF168) recruits but is unable to activate 53BP1 thus creating a barrier to the endorsement of non-homologous end-joining (NHEJ) DNA repair. The simultaneous presence of CtIP facilitates the recruitment of factors essential for engaging the homology-directed repair (HDR) pathway, such as EXO1, hence promoting DNA resection and facilitating the precise repair of the Cas9-induced DSB.