Application of CRISPR-Cas13a System in Downregulation of Zebrafish Genes
XU Mengting, XIAO Wei, LIU Dong*, DUAN Xuchu*
This study aims to establish and validate a CRISPR-Cas13a-based system for downregulating gene expression in zebrafish. In the study, the transgenic reporter gene egfp and the endogenous zebrafish genes ta and fabp11a were selected as target genes for the CRISPR-Cas13a system. Corresponding specific guiding crRNAs and their mutants were designed and synthesized. Cas13a and dCas13a expression vectors were constructed. After in vitro transcription, Cas13a/dCas13a-mRNAs and the guiding crRNAs/crRNA-muts of target genes were co-injected into AB and Tg(β-actin:loxp-egfp-stop-loxp-DTA) zebrafish embryos. Phenotypic observation and qRT-PCR analysis were performed to detect the downregulating effects of Cas13a on the target genes. Following co-injection of Cas13a-mRNA and egfp-crRNA, ta-crRNA, or fabp11a-crRNA respectively, phenotypic observations revealed markedly reduced GFP signals and developmental defects on tails or eyes, which varied among target genes. qRTPCR detection confirmed the reduction of target gene transcripts. CRISPR-Cas13a systems with dCas13a or mutated crRNAs failed to reduce target genes expression. The results indicated that CRISPR-Cas13a system could effectively knock down target gene expression in zebrafish.
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