Abstract
The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to twist and ntl genes. Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish.
Highlights
Engineered animal models play a vital role in the study of genes mutated in human diseases
In order to minimize variables that may have an effect on the outcome of editing, we selected small guide RNA (sgRNA) that have previously been shown to induce C → T or G → A conversion in the zebrafish using BE3 [24]
Our data showed that twist2-T1 (6/8 embryos) and ntl (7/8 embryos) sgRNAs caused indels detected as multiple peaks, whereas twist2-T2 (0/8 embryos) failed to cause indels at detectable levels in our strain of fish (Figure 2A)
Summary
Engineered animal models play a vital role in the study of genes mutated in human diseases. When mutations in a gene are identified in human patients, animal models are developed to prove disease causation (display of similar phenotype), understand disease mechanism and identify potential treatments. Targeted genome editing by CRISPR/Cas technology provides an inexpensive, easy to use, and versatile method to generate gene knockout zebrafish models [3,5]. Several laboratories including ours have established high-throughput methods to generate zebrafish with knockout of single or multiple genes using CRISPR/Cas9 [6,7,8,9,10].
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