敲降（MO/CRISPR） Morpholino knock-down技术原理 图1. Morpholino 技术原理。（a）Morpholino与RNA结合。（b）Morpholino 通过阻断翻译过程而发挥作用。（c-e）Morpholino阻断RNA的正常剪接。 Morpholino Knockdown应用案例 图2. Morpholino Knockdown 靶点位置示意图。The zebrafish gene-X was targeted by three specific morpholino antisense strategies to prevent either the translation of the zebrafish gene (ATG-MO) or proper splicing of exon 3 (E3I3-MO). Morpholino Knockdown表型分析 图例. Panels A through H show lateral views of control MO injected zebrafish embryos (Panel A and Panel E) and embryos injected with gene-X morpholino oligonucleotides (MO) (Panel B through C, Panel F through G), gene-X-e3i3-MO plus nonmutant zebrafish gene-X (Panel D and Panel H). Coinjection of nonmutant zebrafish gene-X mRNA rescued U-shaped somites (red arrow) and curved body axis (blue dotted line) in gene-X morphants at 52 hpf. The bar graph in Panel I shows the percentage of embryos with development defects.Panels J Effectiveness of gene-X knockdown was confirmed by RT-PCR and sanger sequencing. hpf, hours post fertilization. Morpholino Knockdown信号通路机制分析 图例. Endogenous shha, ptch1, ptch2, sufu, gli1, gli2a, gli2b, and gli3 in wild-type control and geneX morphants assessed by qRT-PCR (n = 100 individual embryos). geneX fine-tunes Hedgehog patterning activity maybe through a novel regulatory feedback loop. 基因Knockdown后抑制斑马鱼血管生成 图例. GeneX knock down inhibits the trunk angiogenesis in zebrafish. (A-D) Representative fluorescent images of zebrafish embryos at 32h post-fertilization (hpf). (C-E) Compared with wild-type control, embryos injected with geneX-MO present a lower number of incomplete ISVs and only occasional sprouts (asterisk) of dorsal aorta. The boxed regions are shown at higher magnification in the right panels. DLAV, dorsal longitudinal anastomotic vessels; ISV, intersegmental vessels; DA, dorsal aorta; PCV, posterior cardinal vein. 文献发表 Wang MS, Zhang RW, Su LY, et al. Positive selection rather than relaxation of functional constraint drives the evolution of vision duringchicken domestication. Cell Res. 2016 May;26(5):556-73. 基因Knockdown后导致中枢神经系统特异性细胞凋亡 图例. Morpholino knock down of geneX induces potent CNS-specific apoptosis. Wild-type control embryos and embryos injected with geneX-MO were stained with acridine orange (AO) at 32hpf. Apoptotic cells are visible as black spots, and less bright homogenous black staining is unspecific background staining. (A-B) Uninjected wild-type control zebrafish exhibited few or no apoptotic cells in CNS (central nervous system). In contrast, significantly increased staining was observed throughout the CNS in zebrafish injected with geneX-MO (C-D). The blue boxed regions are shown at higher magnification in the right panels. A-D: lateral view, anterior, left. CRISPRi CRISPRi技术是在CRISPR/Cas9基础上，将Cas9蛋白改造成为不具有切割活性的dCas9，在sgRNA的介导下仍可以靶向基因组序列。CRISPRi技术能够有效的抑制编码基因和非编码基因的表达，并可同时对多个基因进行调控且无脱靶率。CRISPRi技术的出现为研究基因的功能提供了高效的新方法。