Cure Rare Disease, in partnership with Taconic Biosciences, has successfully developed a knock-in mouse model harboring a mutation known to cause ADSSL1 gene-related myopathy. This model will be used in in vivo testing, an essential step in the development of therapeutics for rare and ultra-rare genetic disorders. 

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What is a knock-in mouse model?

When developing mouse models, researchers may choose to develop knock-in or knock-out models. In knock-out models, a specific gene is inactivated in order to analyze the effects that having a nonfunctional gene may have on an organism.

In knock-in models, on the other hand, an existing gene is altered in some way, such as adding in a specific genetic sequence. This method is extremely useful in drug development because by generating a model with a disease-causing mutation, the therapeutic’s efficacy can be further analyzed.

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How was CRD’s ADSSL1 model generated?

CRD’s ADSSL1 mice were produced using CRISPR/Cas9 mediated gene editing. To produce the desired mutations, a Cas9 protein, gRNA, and an oligonucleotide were injected into zygotes. Guide RNA binds itself to a section of DNA, allowing the Cas9 enzyme to cut the DNA at the location indicated by the guide RNA. The oligonucleotide is a short DNA sequence that acts as a template for gene editing. 

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What mutations does this model harbor?

This model contains a point mutation. In this case, a short sequence of DNA nucleotides, GAC, were altered to be AAT. Additionally, a silent mutation was inserted slightly downstream on the exon. Silent mutations are those that do not alter the amino acid coded for. However, by making this alteration, a restriction site is generated. Restriction sites are sequences of nucleotides, often palindromic, that restriction enzymes can bind to and cut to produce DNA fragments. Creating restriction sites is an important tool that can be used for analysis via electrophoresis or polymerase chain reaction.