Showing papers by "Guy-Franck Richard published in 2019"
TL;DR: Different therapeutic approaches using highly specific single- or double-strand endonucleases targeted to trinucleotide repeat loci are compared, and relative efficacies and specificities of these nucleases will be discussed.
Abstract: Trinucleotide repeats are a particular class of microsatellites whose large expansions are responsible for at least two dozen human neurological and developmental disorders Slippage of the two complementary DNA strands during replication, homologous recombination or DNA repair is generally accepted as a mechanism leading to repeat length changes, creating expansions and contractions of the repeat tract The present review focuses on recent developments on double-strand break repair involving trinucleotide repeat tracts Experimental evidences in model organisms show that gene conversion and break-induced replication may lead to large repeat tract expansions, while frequent contractions occur either by single-strand annealing between repeat ends or by gene conversion, triggering near-complete contraction of the repeat tract In the second part of this review, different therapeutic approaches using highly specific single- or double-strand endonucleases targeted to trinucleotide repeat loci are compared Relative efficacies and specificities of these nucleases will be discussed, as well as their potential strengths and weaknesses for possible future gene therapy of these dramatic disorders
29 citations
TL;DR: It is demonstrated that secondary structure formation on the guide RNA was a major determinant of nuclease efficacy, and the most efficient of all CRISPR-Cas nucleases was Streptococcus pyogenes Cas9.
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 gene editing approach. Here, a new assay was developed to test several CRISPR-Cas nucleases on microsatellites involved in human diseases, by measuring at the same time double-strand break 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. All repeat tracts did inhibit double-strand break resection. We demonstrate that secondary structure formation on the guide RNA was a major determinant of nuclease efficacy. Using deep sequencing, off-target mutations were assessed genomewide. Out of 221 CAG/CTG or GAA/TTC trinucleotide repeats of the yeast genome, three were identified as carrying statistically significant low frequency mutations, corresponding to off-target effects.
3 citations
TL;DR: Data show that SpCas9 is probably not a good choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.
Abstract: Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent [~]2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal rearrangements around the repeat tract. These rearrangements depended on RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50{Delta} strain, whereas they were impaired in a sae2{Delta} mutant, only on the DSB end containing most of the repeat tract. This proved that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end.nnIn addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfer with gene conversion too. Altogether, these data show that SpCas9 is probably not a good choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.