ArticleMethods in molecular biology (Clifton, N.J.)2024
Generation of Transgenic Fish Harboring CRISPR/Cas9-Mediated Somatic Mutations Via a tRNA-Based Multiplex sgRNA Expression.
Article in Methods in molecular biology (Clifton, N.J.), 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
The controlled expression of Cas9 and/or sgRNA in transgenic zebrafish made it possible to knock out a gene in a spatially and/or temporally controlled manner. This transgenic approach can be more useful if multiple sgRNAs are efficiently expressed since we can improve the biallelic frame-shift mutation rate and circumvent the functional redundancy of genes and genetic compensation. We developed the tRNA-based system to express multiple functional sgRNAs from a single transcript in zebrafish and found that it is applicable to the transgenic expression of multiple sgRNAs. In this chapter, we describe a procedure for the generation of plasmids containing multiple sgRNAs flanked by tRNAs and a method to induce multiple CRISPR/Cas9-mediated genome modifications in transgenic zebrafish.
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