Genetic engineering a large animal model of human hypophosphatasia in sheep

Abstract

The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.

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.…”

“…They combined CRISPR with SCNT to obtain CFTR e/e lambs with a similar phenotype as in humans, providing an interesting model to advance the development of new therapies. 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].…”

“…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].…”

Testes of DAZL null sheep lack spermatogonia and maintain normal somatic cells

“…Our data provide another proof-of-principle for using HDR to precisely modify the sheep genome 28 . This has been shown to be useful for producing human disease models in areas where sheep more physiologically relevant 28 . The HDR approach was convenient as the appearance of a known allele simplifies the screening of strains.…”