Clonal analysis of gene loss of function and tissue-specific gene deletion in zebrafish via CRISPR/Cas9 technology.

In the last few years the development of CRISPR/Cas 9-mediated genome editing techniques has allowed the efficient generation of loss-of-function alleles in several model organisms including zebrafish. However, these methods are mainly devoted to target-specific genomic loci leading to the creation of constitutive knock-out models. On the contrary, the analysis of gene function via tissue- or cell-specific mutagenesis remains challenging in zebrafish. To circumvent this limitation, we present here a simple and versatile protocol to achieve tissue-specific gene disruption based on the Cas9 expression under the control of the Gal4/upstream activating sequence binary system. In our method, we couple Cas9 with green fluorescent protein or Cre reporter gene expression. This strategy allows us to induce somatic mutations in genetically labeled cell clones or single cells, and to follow them in vivo via reporter gene expression. Importantly, because none of the tools that we present here are restricted to zebrafish, similar approaches are readily applicable in virtually any organism where transgenesis and DNA injection are feasible.

De Santis F ，Di Donato V ，Del Bene F 《Methods in Cell Biology》

Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish.

Yin L ，Maddison LA ，Chen W 《Methods in Cell Biology》

CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.

Luo JJ ，Bian WP ，Liu Y ，Huang HY ，Yin Q ，Yang XJ ，Pei DS ... -  《-》

CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish.

Jaenisch R ，Zhang F ，Gage F ，Albadri S ，De Santis F ，Di Donato V ，Del Bene F ... -  《-》

Zebrafish Genome Engineering Using the CRISPR-Cas9 System.

Geneticists have long sought the ability to manipulate vertebrate genomes by directly altering the information encoded in specific genes. The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease has the ability to bind single loci within vertebrate genomes and generate double-strand breaks (DSBs) at those sites. These DSBs induce an endogenous DSB repair response that results in small insertions or deletions at the targeted site. Alternatively, a template can be supplied, in which case homology-directed repair results in the generation of engineered alleles at the break site. These changes alter the function of the targeted gene facilitating the analysis of gene function. This tool has been widely adopted in the zebrafish model; we discuss the development of this system in the zebrafish and how it can be manipulated to facilitate genome engineering.

Li M ，Zhao L ，Page-McCaw PS ，Chen W ... -  《-》