The invention discloses a method for directional knockdown of a multi-copy
gene in
zebrafish genome. The method comprises the following steps: I, preparing a
zebrafish embryo; II, constructing an
expression vector of dCas9-Eve
fusion protein; III, conducting in-vitro transcription of sgRNA, dCas9-Eve, Cas9 and
gene, and purifying the sgRNA, dCas9-Eve, Cas9mRNA and
gene mRNA, which are synthesized through the in-vitro transcription, by virtue of RNeasy Mini Kit; IV, identifying activity of the sgRNA; and V, knocking out expression of the multi-copy gene znfl1s by virtue of a dCas9-Eve method, and judging the situation that the
gene expression is knocked out by detecting the expression of the gene by virtue of an
embryo whole-
mount in situ hybridization method. The method provided by the invention, by virtue of the dCas9-Eve, can specifically knock down the transcription of the gene under the circumstance of implementing the effective targeted identification of sgRNA existence of a gene
promoter, so that the researched gene is prevented from being affected by MO
toxicity or non-specific
phenotype caused by target missing; and moreover, since an Eve inhibiting transcription structural domain is derived from
zebrafish, the dCas9-Eve is applicable to knockdown of any zebrafish genes at a transcription level.