Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Modeling Craniosynostosis in Zebrafish using the Genome Editing Technique CRISPR
Tatiana Favelevic, B.S. Pending1, Rebecca Anderson, Ph.D. Candidate2, Jacek Topczewski, Ph.D. Candidate1, Arun K. Gosain, M.D.3, Jolanta M. Topczewska, Ph.D.1.
1Ann & Robert H. Lurie Children's Hospital of Chicago Research Center, Chicago, IL, USA, 2Northwestern University, Chicago, IL, USA, 3Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA.

Craniosynostosis (CS), or premature fusion of the cranial bones, represents a clinically and genetically heterogeneous illness, with multifactorial etiology. More than a hundred mutations within the TWIST1 gene are associated with autosomal dominant disorders of CS in humans, a consequence of TWIST1 haploinsufficiency. Moreover, Twist1 heterozygote mice develop similar defects as humans, including coronal suture synostosis. To test the feasibility of a zebrafish model for the study of CS and to assess the degree of similarity between rodents and fish in the molecular mechanisms that control cranium development, we have employed new technology of genome editing (CRISPR/Cas) to mutate the twist homologue. We selected zebrafish twist3 based on strong expression in the suture mesenchyme as revealed by in situ RNA hybridization. The CRISPR/Cas system uses CRISPR guided RNA (gRNA) working in a complex with Cas endonucleases to target and cleave target DNA. When a double stranded break is introduced into the target DNA, a non-homologous end joining repair mechanism creates a mutation. We anticipate that mutagenesis of twist3 using CRISPR technology will result in a novel zebrafish model of craniosynostosis. Moreover, it will provide us with the possibility of rapid genetic manipulations of CS related genes to better understand the molecular mechanisms controlling normal and pathological development of cranial sutures.
METHODS:
The CRISPR-twist3 construct was created by PCR method, the gRNA, transcribed in vitro, and all constructs including cas9 mRNA were injected into zebrafish embryos. For diagnostic reasons, the CRISPR/Cas cleavage site was designed next to a MspI restriction site, permitting easy identification of genome editing events through the creation of restriction enzyme polymorphisms. This diagnostic digestion will be used for genotyping of the progeny of injected fish when testing for the inheritance of the twist3 mutation. CRISPRtwist3 injected fish, aged 2-3 months, will be out crossed with wild type. The germ line transmission of the twist3 mutation would ensure a stable line. We predict that 3-5 founder fish will be isolated. The twist3 gene will be sequenced to define the mutation.
RESULTS:
Randomly selected, CRISPR/Cas RNAs-injected and non-injected embryos were genotyped as described above. We detected incomplete digestion of PCR product for ~30% of the injected embryos (n=76), while the controls were fully digested by MspI, indicating that 30% of injected fish may carry the mutation within twist3. We expect this is sufficient to recover stable mutant strain in the next generation.
CONCLUSION:
The present study demonstrates the effectiveness of the CRISPR/Cas method in editing the zebrafish genome. We predict that within 6 months, stable mutant strains can be established, providing the possibility of examining genetic interaction among genes and signaling pathways.


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