CRISPR/Cas9 mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease)

Abstract

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 ( PPT1 ) gene and severely reduces the child’s lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.

CRISPR in livestock: From editing to printing

“…If an incompatible PAM or target site sequence is introduced into the genome via the knock-in, the sgRNA-Cas9 complex can no longer bind to the target site, and no further edits can be made. The use of ssODN to induce knock-in or to improve knockout efficiency has been reported recently in ruminant species (sheep: [15,16]; goats: [17]; cows [27]).…”

“…When SCNT is mastered by the laboratory, the low efficient rate will be compensated with most of the healthy born animals carrying the desired mutation. Genome editing mediated by SCNT is applied by some laboratories in some kind of projects (e.g., multiplex editing), but the high efficiency of CRISPR after direct microinjection into zygotes has allowed an easier approach (sheep: [6,7,15,16]; goats: [9,17]; pigs: [11,13]). All together, these reports showed that with CRISPR it is possible to perform direct microinjection into the zygote of different species, with minimum effects on developmental competence and pregnancy rates, high newborn survival rate, and high editing rate with acceptable homozygous proportion.…”

“…Another sheep model of a rare human bone disease, hypophosphatasia, was recently reported using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene, which induced the same disorder as in humans [15]. In addition, an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease), a devastating neurodegenerative pediatric disorder with no cure, was achieved more recently using CRISPR/Cas9 system to introduce the same human mutation [16]. The availability of CRISPR to accurately replicate the clinical phenotype of human diseases in large animals is an invaluable tool for the understanding of disease progression and the development of more effective therapeutics.…”

“…Genome editing conducted by this mechanism can be used to insert a predefined single nucleotide or sequence, or even change or delete it in existing genes. After the first knockouts births reported by NHEJ, successful generation of knock-in large animals by HDR were achieved in sheep [15,16], goats [17] and pigs [18].…”

Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair

“…Although reported in other species [17][18][19] , current literature is scarce for CRISPR assisted ssODN-mediated homologous recombination in ruminant species (i.e., sheep, goats and cattle). Recently, Williams et al 5 reported for the first time the effectiveness of this tool to induce a point mutation in sheep, and Eaton et al 20 described the generation of homozygotes lambs by the insertion of a human mutation into the orthologous sheep locus. In the current study, we successfully generated both indel mutations and HDR-mediated KI using the ssODN strategy in sheep.…”