Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations

Lucie Poggi (1, 2, 3) , Lisa Emmenegger (1) , Stéphane Descorps-Declère (1, 4) , Bruno Dumas (3) , Guy-Franck Richard (1, 2)

1 Dynamique du Génome - Dynamics of the genome
2 Collège Doctoral
3 Sanofi [Vitry-sur-Seine]
4 Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB

Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR-Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG) 33. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.

Domaines: Biochimie, Biologie Moléculaire

Lucie Poggi, Lisa Emmenegger, Stéphane Descorps-Declère, Bruno Dumas, Guy-Franck Richard. Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations. Nucleic Acids Research, 2021, 49 (14), pp.8120 - 8134. ⟨10.1093/nar/gkab569⟩. ⟨hal-03353648⟩