Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis

CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish.CRISPR/Cas9目前已被常规应用于多种研究体系的靶向诱变。本研究报道了利用核酶（ribozyme）在体外及斑马鱼胚胎中合成向导RNA（guide RNA，gRNA）的方法。研究证实，整合核酶序列可在不降低诱变效率的前提下，拓展可用于诱变的启动子类型与靶位点数量。我们通过对比含与不含核酶序列的gRNA构建体的诱变效率验证了上述结论，并利用热激启动子（依赖RNA聚合酶II的启动子）构建了可表达gRNA的转基因斑马鱼，该模型能够诱导其靶位点发生诱变。本方法不仅为gRNA效率检测提供了简化实验方案，同时也提升了斑马鱼条件性基因敲除实验的通用性。