Designing and Construction of Donor DNA for RNA-Guided Recombinase (RGR) Platform-Mediated Knockout of Leptin B (Lepb) Gene in Zebrafish

The RNA-guided recombinase (RGR) platform, combining a hyper-activated sin recombinase with catalytically inactive Cas9 (dCas9), offers a programmable, cell-independent and potentially safer alternative to CRISPR/Cas9 for targeted genome editing. This study was conducted to design and construct a donor DNA plasmid enabling targeted knockout of the lepb gene in zebrafish (Danio rerio) using the RGR platform.

The lepb sequence was retrieved from the NCBI database and sin recombinase-based RGR recognition motifs were identified and analyzed functionally and structurally using CYC_REC, SOPMA and NetSurfP-3.0. SWISS-MODEL generated 3D structures of lepb, which were validated for quality. An experiment was conducted to build a donor DNA plasmid containing a mutated lepb fragment through site-directed mutagenesis (SDM) and verified by sequencing.

A nonsense mutation (GAG to TAG) was introduced at 1738 bp, disrupting a critical glutamate residue, leading to loss of lepb protein function. In silico analysis revealed the loss of key structural elements (partial helix-C, complete helix-CD loop and helix-D residues) and disulfide bridge-forming cysteine residues (C112 and C157), which are crucial for lepb-lepr binding, stability and folding of the lepb protein. The homology modeling confirmed substantial alteration in the lepb conformation due to truncation. The donor DNA plasmid containing a 1808 bp-long lepb fragment with the targeted mutation (T) was constructed and validated. No off-target effects were detected. This study demonstrates the precision and feasibility of RGR-mediated lepb knockout and provides a validated donor DNA plasmid with potential applications in growth trait enhancement and functional genomics in zebrafish.