上海海洋大学学报2025，Vol.34Issue(6)：1193-1202,10.DOI:10.12024/jsou.20241104689
CRISPR-Cas13d系统下调青鳉tyr基因表达的研究
Application of Cas13d system in the down-regulation of tyr gene expression in medaka(Oryzias latipes)
摘要
Abstract
The CRISPR-Cas13d protein targets RNA substrates through crRNA-mediated sequence-specific binding and achieves precise cleavage via its ribonuclease activity,offering a non-genome-editing strategy for RNA-level gene regulation.In this study,we optimized the Cas13d gene sequence based on the codon preference of medaka(Oryzias latipes),successfully constructed a prokaryotic expression system,and purified recombinant Cas13d protein with a molecular weight of 115 ku.By microinjecting in vitro-transcribed Cas13d mRNA complexed with crRNA targeting the tyrosinase gene(tyr)into medaka embryos,alongside parallel experiments using Cas13d protein/crRNA ribonucleoprotein(RNP)complexes,we systematically evaluated RNA-editing efficiency.qRT-PCR and sequencing analyses demonstrated that both delivery methods significantly reduced tyr RNA levels in embryos(P<0.01),while DNA sequencing confirmed the absence of mutations in the target DNA sequences.This study validates the effectiveness and specificity of Cas13d-mediated gene silencing through RNA editing in medaka,not only providing a novel regulatory tool for fish genetic research but also establishing a theoretical foundation for developing RNA-targeted Cas13d antiviral transgenic fish lines.关键词
青鳉/CRISPR-Cas13d/tyr/蛋白纯化/RNA编辑Key words
medaka/CRISPR-Cas13d/tyr/protein purification/RNA editing分类
农业科技引用本文复制引用
许朝然,夏必琳,蒋月雯,邓菊,陈若雪,梁晶婕,陈天圣..CRISPR-Cas13d系统下调青鳉tyr基因表达的研究[J].上海海洋大学学报,2025,34(6):1193-1202,10.基金项目
国家自然科学基金(32273127,31771648) （32273127,31771648）
集美大学科研基金项目(ZQ2020003) （ZQ2020003）
