RF17 | PMON22 KNOCKOUT AND KNOCKIN GENE EDITING BY CRISPR CAS9 IN ZEBRAFISH (DANIO RERIO) AS A GENOTYPE-PHENOTYPE CORRELATION TOOL FOR EXOME-IDENTIFIED GENES IN PATIENTS WITH CONGENITAL HYPOPITUTARISM
Authors: Carvalho Luciani R , Ventura Fernandes Bianca 
Published: November 2022

Abstract

Introduction
Zebrafish has been used as an animal model to study human disease due to its 75% genetic homology with humans besides easier maintenance, small size and high reproductive rate. knockout Gene through CRISPR/Cas genomic edition can lead to an inactivating protein due to frameshift and stop codon, while Knockin allows a specific base edition. Whole exome sequence allows the identification of new/rare variants as molecular diagnosis in patients with congenital hypopituitarism (CH), although sometimes phenotype-genotype is mandatory and edited zebrafish genome can be a tool. The aims of this work was to generate the gh1 knockout gene in zebrafish, mimetizing a patient with CH harboring GH1 c.171delT (p.Phe57Leufs*43) allelic variant leading to frameshift and stop codon and cdh2 knockin due to CDH2 c.865G&gt;A (p.Val289Ile) allelic variant in homozygosity in a patient with CH.

Methods
For the gh1 knockout gene 2 different guides (sgRNA) were designed in exon 5. At one cell stage a total number of 274 embryos were injected with different sgRNA and Cas9. At 3 months after fertilization, 12 animals had their tail DNA extracted followed by PCR amplification with primers flanking exon 5 and the product was Sanger sequenced. For the cdh2 knockin g&gt;a for p.Val289Ile, base editing plasmid was used, containing an inserted DNA with cytidine deaminase fused to Cas9 nickase (nCas9), that direct convert C to T or G to A in the Zebrafish DNA sequence of interest. Twenty-four hours after injection, a total of 100 embryo's DNA were collected and extracted followed by PCR amplification and Sanger sequenced in order to check mutagenesis efficiency.

Results
For the gh1 knockout gene animals that reached sexual maturity, two of them with an allelic variant leading to frameshift and stop codon (animal 1 harboring ENSDARG00000038185: g.568_572del, animal 2 harboring ENSDARG00000038185: g.554_560insACGAACG) were prioritized for the strains construction. These animals (F0) were mated with WT animals and their offspring (F1) were genotyped, showing mutations not seen in the tail sequencing of the parents, suggesting a gonadal mosaic in F0 generation. At 3 months these animals (F1) were mated and F2 offspring with pure heterozygous mutation were generated and crossed with WT in order to obtain the mendelian rate of WT, HT and Homo to characterize the animal phenotype. From 100 animals injected for cdh2 knockin approach, 10 random animals have already been sequenced and presented no expected pathogenic allelic variant.

Conclusion
The genomic editing technique by Crispr cas was efficient to generate a pure heterozygous gh1 knockout animal in order to obtain homozygous animal in F3 for phenotype characterization, while knockin was inefficient for the analyzed number of eggs although it is missing 90 animal to be analyzed in order to calculate the real technique efficiency.
Presentation: Sunday, June 12, 2022 12:30 p.m. - 12:35 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.