Engineering precision zebrafish alleles of human disease

Area of Science:

• Genetics and Genomics
• Developmental Biology
• Biotechnology

Background:

• Animal models are vital for understanding human disease pathogenesis and evaluating therapies.
• Human patient genome sequencing reveals unique variants causing distinct disease phenotypes.
• The UAB Center for Precision Animal Modeling (CPAM) focuses on analyzing patient variant pathogenicity and disease mechanisms via animal models.

Purpose of the Study:

• To optimize a zebrafish gene editing platform for generating animal models of human patient variants.
• To evaluate the efficiency of different CRISPR/Cas9 and single-stranded oligodeoxynucleotide (ssODN) parameters for homology directed repair (HDR).
• To establish reliable methods for identifying germline transmitting founders and genotyping F1 animals.

Main Methods:

• Utilized CRISPR/Cas9 gene editing with ssODN repair templates in zebrafish to generate 11 patient variants and 1 research allele.
• Evaluated multiple oligo orientations and sizes, and employed PCR amplicon Next Generation Sequencing (NGS) for F0 embryo HDR efficiency assessment.
• Used NGS on F0 progeny DNA to identify germline transmitting founders and High-Resolution Melting Curve Analysis (HRMA) for F1 genotyping.

Main Results:

• Successfully generated 11 patient variants and 1 research allele in zebrafish.
• Determined that empirical evaluation of guide and oligo combinations is necessary for optimal HDR rates, with NGS being superior to ICE for frequency determination.
• Observed a 'jackpot' effect in germline transmission in most founders, and confirmed HRMA's utility in differentiating F1 animals with patient variants, while noting potential false positives with other genotyping methods.

Conclusions:

• The optimized zebrafish gene editing platform effectively generates animal models for human patient variants.
• NGS is a reliable method for assessing HDR efficiency and identifying germline transmitting founders.
• HRMA provides a robust method for genotyping F1 animals carrying patient variants.