Targeted ingression of SNPs into the zebrafish genome - Despite recent advances in gene editing, the ability to make small changes in the genomes of model organisms remains a challenge. In zebrafish, enhanced oligo design, base-editing and prime-editing show promise to increase the frequencies of small changes but their application is still cumbersome due to low germline transmission rates, requiring extensive and time-consuming screening. This bottleneck prevents the efficient targeted production single nucleotide polymorphisms (SNPs) for disease modeling, limits our ability to perform mechanistic structure-function studies, and prevents rigorous determination and the assessment of variants of uncertain significance (VUS) that promote human disease. The overarching goal of the proposed experiments is to create a gene editing method to efficiently introduce SNPs and VAPs into the zebrafish genome for modeling human health. We have used short regions of homology surrounding a CRISPR/Cas9 double strand breaks to efficiently target integration of DNA cargos for mutagenesis, protein tagging, Gal4 reporters, Cre-drivers and conditional alleles, using a method we have coined GeneWeld. Key to this methodology is repair at a double strand break in genome produced by injection of Cas9 mRNA and a genomic gRNA with a plasmid template. The circular plasmid template is cut in vivo with a gRNA, called a universal gRNA (3), that recognizes sequences adjacent to the short homology to produce a linear template for integration. The repair and subsequent integration of the cargo is likely utilizing microhomology-mediated end joining (MMEJ) or a single-strand annealing (SSA) mechanism and results in germline transmission of the targeted integration events in approximately 50% of the F0 injected embryos to the next generation. Since this methodology remains more efficient that other strategies for targeted CRISPR/Cas9-targeted integration in zebrafish, we propose that GeneWeld can be used to efficiently target ingression of SNPs and VUS into the zebrafish genome. For this, we will test the ability of GeneWeld to introduce SNPs at three different loci. We will also examine whether co-selection of RFP repair correlates with ingression of small changes in the genome at a separate gene. Additionally, we will enhance the ability to generate SNPs and VUS in zebrafish by overexpression of DNA repair enzymes and dominant mutations in repair enzymes. Key preliminary data indicates that overexpression of a variant of Rad51 reproducibly enhances repair. We will use this approach to examine the activity of Rad51 for the ingression of small nucleotide changes. The proposed experiments are expected to develop simple and efficient methods to target SNPs and VUS into any gene in the zebrafish genome. In addition, we will generate an important model with a dead RFP knock-in that will be of great utility to assess editing and may provide a means for co-selection of editing events. Based on the ability of GeneWeld to be used in other organisms, we expect these methodologies to be broadly applicable to animal models used by the NIH.
