Plastic Surgery Research Council
Members Only  |  Contact  |  PSRC on Facebook
PSRC 60th Annual Meeting
Program and Abstracts

Back to 2015 Annual Meeting Program


Modeling Pfeiffer Syndrome in zebrafish by CRISPR-targeted homologous recombination
Joanna P. Tomaszewski, MS, Arun K. Gosain, MD, Jolanta M. Topczewska, PhD.
Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA.

PURPOSE:
Fgf/Fgfr signaling plays an essential role in endochondral and intramembranous ossification. The Pro252Arg gain-of-function substitution in FGFR1 is associated with Pfeiffer syndrome in humans, and leads to craniosynostosis (CS). Utilizing CRISPR/Cas9 genome editing in combination with homologous recombination, the present study aims to mimic the Pro252Arg mutation within the fgfr1a zebrafish locus. In addition to creating a novel CS model bearing many advantages over existing mammalian models, we aim to establish a novel method that allows for nucleotide substitutions within a targeted locus.
METHODS:
CRISPR/Cas9 genome editing utilizes guide RNA (gRNA) to target a specific DNA sequence and Cas9 endonuclease to create a double stranded break at the target site. Upon cleavage, a non-homologous end joining repair mechanism is induced by the cell to fix damaged DNA. In our novel approach we designed an exogenous site-specific oligonucleotide template, which bears the mutated sequence. When provided together with gRNA and cas9 mRNA, the cas9 targeted fgfr1 region is substituted with the desired Pro252Arg mutation.
Construct Design: The CRISPR-fgfr1a DNA construct was designed based on the genomic fgfr1a sequence. The corresponding gRNA-fgfr1a was transcribed in vitro. An oligonucleotide containing the mutated sequence flanked by homologous recombination arms was designed and manufactured.
Delivery of CRISPR-fgfr1a: The gRNA, cas9 mRNA, and oligonucleotide were coinjected into single-cell stage embryos.
Genotyping: To identify genome-editing event, specific primers were designed to amplify the modified sequence by PCR. A unique 542 bp fragment is only amplified when tested DNA carries the Pro252Arg substitution. Furthermore, a diagnostic restriction site for EagI is lost in the PCR product when successful recombination occurs. Adult CRISPR-fgfr1a injected fish were outcrossed with WT fish to determine germline transmission.
RESULTS:
Injected G0 fish (n=41) were genotyped as described above. We detected 542 bp fragments in addition to incomplete digestion of DNA for ~80% of injected fish (n=32), indicating high efficiency of the CRISPR/Cas9-mediated mutagenesis. WT controls were fully digested by EagI. We observed phenotypic differences of varying degree in G0 fish, including dysmorphic head shape and abnormal scale patterning, consistent with fgfr1 misregulation (Figure 1). These features were also observed in the progeny, which are currently being genotyped to confirm germline transmission of Pro252Arg. The effects of genetic manipulation on suture phenotype will be assessed through histological analysis, skeletal morphology, and gene expression studies.
CONCLUSION:
We demonstrate the effectiveness of CRISPR/Cas9- targeted homologous recombination in editing the zebrafish genome. Preliminary assessment suggests that Fgfr1-mutant fish exhibit a phenotype consistent with fgfr1 misregulation.
Once validated, the proposed zebrafish model of Pfeiffer syndrome would provide a new tool to further assess the role of Fgf signaling in cranial suture pathology. Moreover, the CRISPR/Cas9/recombinant oligo method allows us to efficiently manipulate other genes in the pathway to better understand molecular mechanisms controlling normal and pathological development of cranial sutures.


Back to 2015 Annual Meeting Program