Microsatellites Involved in Human Disorders are Differentially Cut by CAS Nucleases Due to Secondary Structure Formation

Abstract

Microsatellite expansions are the cause of more than 20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a therapeutic approach. Here, an in vivo biochemistry assay was designed to test several CRISPR-Cas nucleases on microsatellites involved in human diseases, by measuring at the same time double-strand break (DSB) rates, DNA end resection and homologous recombination efficacy. Broad variations in nuclease performances were detected on all repeat tracts. Streptococcus pyogenes Cas9 was the most efficient of all and Staphylococcus aureus Cas9 the least one. Cas12a was the most active on GAA trinucleotide repeats. All repeat tracts did inhibit DSB resection. We demonstrate that neither gRNA or protein levels, nor DSB resection were strongly impacting nuclease efficacy on repeat tracts, the limiting step of the reaction being secondary structure formation on the guide RNA.