Allele-specific CRISPR/Cas9 cleavage approach for two epidermolysis bullosa simplex mutations

Mbarka Bchetnia^1,2, Julie Powell³, Charles Morin^1,4, Catherine McCuaig³, Audrey Dupérée⁴, Jacques P. Tremblay⁵, Catherine Laprise^1,2

¹Université du Québec à Chicoutimi, Département des sciences fondamentales, Saguenay, Canada;
²Université du Québec à Chicoutimi, Centre intersectoriel en santé durable, Saguenay, Canada;
³Hôpital Ste-Justine, Montréal, Canada;
⁴Centre intégré universitaire de santé et des services sociaux du Saguenay-Lac-St-Jean, Saguenay, Canada;
⁵Centre de recherche du CHU de Québec, Université Laval, Québec, Canada;

Epidermolysis bullosa simplex (EBS) is a rare genetic disorder of the skin causedby dominant-negative mutations in either keratin 5 (KRT5) or keratin 14 (KRT14) resulting in impairment of keratin filament network and skin bubbles formation. More than 230 EBS causative mutations were described.Until now, there is notreatment for EBS and the care is primarily palliative. The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) system, eight years ago, raised hope for the treatment of EBS by mutant allele-specific gene disruption induced by non-homologous end-joining (NHEJ).

Here, we aim to disrupt the mutant allele for two heterozygous EBS missense mutations in KRT5. Both mutations generate a novel protospacer-adjacent motif (PAM) for the endonuclease Streptococcus pyogenes Cas9 (SpCas9). Thus, for each mutation, we designed a guide RNA sequence (sgRNA) that guides the Cas9 to introduce a DNA cleavage only within the mutant allele in patient’s primary cells.

We achieved successful stringent mutant allele specific knockout indicating permanent mutant allele-specific inactivation. The wild-type allele remains unaffected and its protein product should be sufficient for normal function. Our next step is using this approach in induced pluripotent stem cells (iPSCs) generated for the two patients and then differentiate them into keratinocytes. Gene repaired keratinocytes will serve to engineer skin tissue that will be transplanted to patients as autografts.

Our study describes allele specific CRISPR/Cas9 gene inactivation at a novel PAM created by two EBS mutations and provides a model for application of such strategy to other dominantly inherited diseases.