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Identifying New Mutations Of Fgfr1a After CRISPR/Cas9 Genome Editing
Lauren Kelsey1, Joanna K. Ledwon, PhD1, Jacek Topczewski, PhD2, Arun K. Gosain, MD3, Jolanta M. Topczewska, PhD1.
1Northwestern University Feinberg School of Medicine, Department of Surgery Plastic Division, Stanley Manne Children's Research Institute, Chicago, IL, 2Northwestern University Feinberg School of Medicine, Department of Pediatrics, Chicago, IL, 3Division of Pediatric Plastic Surgery, Ann and Robert H. Lurie Children's Hospital of Chicago and Northwestern University Feinberg School of Medicine, Chicago, IL.

Mutations in FGF receptor 1 are associated with numerous inherited skeletal disorders. We aim to develop a new zebrafish model of fgfr1a deficiency to study molecular mechanisms causing abnormal skeletogenesis. We used CRISPR/Cas9 genome editing method, that provides the ability to introduce heritable mutations in the gene encoding of the protein of interest. Here we aim to characterize an array of mutations within fgfr1a, generated by CRISPR/Cas9 technology ultimately to assess the effect of each mutation on cranium development.
A CRISPR-fgfr1a constructs were used to edit the fgfr1a gene close to the P252R position frequently mutated in patients with Pfeiffer syndrome. Each identified allele was recovered as heterozygote and used for breading to obtain next generation of homozygote mutants. For genotyping, the genomic DNA was isolated from a small fragment of zebrafish caudal fin. Two different sets of primers were designed for PCR amplification of the targeted DNA. The product size was evaluated by electrophoresis on 3.5% MetaPhor agarose gel and selected samples were Sanger sequenced for conformation.
Nine different lines were initially characterized by allelic changes in the genomic sequence. The following mutations were recovered: three frameshift caused by deletion of 20 base pair (bp), 17 bp and 13 bp. These mutations cause changes in the reading frame and the premature termination of protein synthesis, which presumably leads to loss of fgfr1a function. In addition short, in-frame insertion of 6bp and deletion of 3bp were recovered. In this category, we also identified a small, in-frame rearrangement of genomic sequence, which consists of small deletion and substitution within the targeted region (Table 1 and Fig 1). No mutations have been found in fgfr2 or fgfr3 in any of the identified mutant lines.

The CRISPR/Cas9 method targeted to a highly conserved sequence of fgfr1a introduced a diverse array of mutagenized alleles providing the opportunity to investigate suture development in correlation with new pathologic alleles. These mutations are heritable and despite the high sequence homology among genes encoding Fgf receptors, no off-target mutations have been identified to date. Initial characterization of identified alleles will focus on embryonic stages to assess the effect of mutation on Fgf signaling target genes. This analysis will be complemented by histology of zebrafish head to assess the cranial suture development and maintenance of juvenile and adult animals.

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