Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

By Nannan Chang, Changhong Sun, Lu Gao, Dan Zhu, Xiufei Xu, Xiaojun Zhu, Jing Xiong, J. Xi. Cell Research, 2013

810 citations recorded by the source corpus.

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrpy11 mutant embryos or cardia bifida phenotypes in fautm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/60f43df4e21d9400ff7f922612944fdd6f9170ba

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Citations in the corpus

Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. (2013, background): “Recently, several groups have demonstrated the use of the CRISPR/Cas9 system for genome engineering in various species (Chang et al., 2013; Cho et al., 2013; Cong et al., 2013; Friedland et al., 2013; Hou et al., 2013; Hwang et al., 2013; Jinek et al., 2013; Li et al., 2013; Mali et al., 2013; Nekra”
Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system (2013, methodology): “The mutagenesis efficiencies in these two studies are ∼24–60% (15) and ∼35% (16), respectively.”
RNA-guided genome editing in plants using a CRISPR-Cas system. (2013, neutral): “, 2013), zebrafish (Chang et al., 2013; Hwang et al., 2013), yeast (Dicarlo et al.”
Zebrafish: Development of a Vertebrate Model Organism (2018, positive): “Hence many labs have recently demonstrated highly efficient genome editing in zebrafish with this system (Chang et al., 2013; Hwang et al., 2013; Jao, Wente, & Chen, 2013; Li, Zhao, Page-McCaw, & Chen, 2016; Ota, Hisano, Ikawa, & Kawahara, 2014; Varshney et al., 2015).”
Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease (2013, background): “E-mail: oconnorgiles@wisc.edu Genetics, Vol. 194, 1029–1035 August 2013 1029 eukaryotic genomes, including cultured mammalian cells, human stem cells, and yeast (Cho et al. 2013; Cong et al. 2013; Dicarlo et al. 2013; Ding et al. 2013; Jinek et al. 2013; Mali et al. 2013).”
Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018, background): “An alternative option is to use Cas9 mRNA together with sgRNA [27, 92].”
CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes (2015, background): “…have lent itself to a variety of applications, including genome editing, gene function investigation, and gene therapy in animals and human cells (Chang et al., 2013; Cho et al., 2013; Cong et al., 2013; Friedland et al., 2013; Hsu et al., 2014; Hwang et al., 2013; Ikmi et al., 2014; Irion et…”