In biological studies, insertion of exogenous sequences, such as protein tags, fluorescent reporters or loxp sites into targeted genomic locus provides a powerful tool to investigate the spatial-temporal expression patterns of a gene and its functions of interest. To achieve this goal in zebrafish, the engineered endonucleases (EENs), such as TALENs or CRISPR/Cas9 are utilized, as the first step to generate double strand breaks (DSBs). Subsequently, while majority of these DSBs are repaired by non-homology end joining (NHEJ) pathway that generates small deletion or insertion, a small number of them, if provided with donor plasmid containing homologous arms, could also be repaired by homologous recombination (HR) that results in the precise replacement of endogenous DNA fragment...[ Read more ]

In biological studies, insertion of exogenous sequences, such as protein tags, fluorescent reporters or loxp sites into targeted genomic locus provides a powerful tool to investigate the spatial-temporal expression patterns of a gene and its functions of interest. To achieve this goal in zebrafish, the engineered endonucleases (EENs), such as TALENs or CRISPR/Cas9 are utilized, as the first step to generate double strand breaks (DSBs). Subsequently, while majority of these DSBs are repaired by non-homology end joining (NHEJ) pathway that generates small deletion or insertion, a small number of them, if provided with donor plasmid containing homologous arms, could also be repaired by homologous recombination (HR) that results in the precise replacement of endogenous DNA fragment by the sequence of interest. Since HR-mediated knock-in methods occur at such a low frequency (~1%), the CRISPR/Cas9 system has been further modified to break the genomic locus and donor plasmid simultaneously. This concurrent cleavage eventually leads to the direct ligation of genomic DNA and donor sequence through non-homology end joining (NHEJ) pathway at high efficiency (20~40%).

Based on these principles for developing knock-in methods, it’s further modified to achieve visible conditional knock-out in zebrafish. Targeting at Tyrosine hydroxylase (th) for its high efficient sgRNA target, high expression level and identifiable expression pattern, the newly developed visible conditional knock-out methods were proved feasible for further studying target genes expression patterns and their functions in temporal-spatial resolution.