Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

Abstract

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.

NudC regulates actin dynamics and ciliogenesis by stabilizing cofilin 1

“…A recent study showed that generation of robust biallelic mutations by CRISPR/ Cas9 system in the injected zebrafish embryos appears to allow for phenotypic analysis directly in the F0 animals [27]. To further confirm the function of NudC during zebrafish development, we designed a guide RNA (gRNA) to target zebrafish NudC.…”

Heritable gene-targeting with gRNA/Cas9 in rats

“…Technologies of ZFN and TALEN have made genetargeting in the rat genome more convenient and practical [7][8][9]. The CRISPR/Cas system is the most recently developed technology for targeted genome modification in mammalian cells, bacteria, zebrafish and mice [10][11][12]. This system requires a locus-specific CRISPR RNA (crRNA), a transactivating crRNA (tracrRNA) and a nuclease Cas9.…”

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…Over 35% of site-specific mutations were found when specific Cas/gRNA was used to target genes in living zebrafish embryos. The study also successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos, and showed that the Cas9/gRNA system has the potential to be a simple and efficient reverse genetic tool for zebrafish and other model organisms [20].…”