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© Research
Publication : Nucleic acids research

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

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Nucleic acids research - 20 Aug 2021

Poggi L, Emmenegger L, Descorps-Declère S, Dumas B, Richard GF,

Link to Pubmed [PMID] – 34233005

Link to DOI – 10.1093/nar/gkab569

Nucleic Acids Res 2021 08; 49(14): 8120-8134

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.