Abstract
Recent use of the CRISPR/Cas9 system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis. Here we developed a simple, highly efficient, and large-scale genome editing method, in which the RNAs for the CRISPR/Cas9 system are electroporated into zygotes rather than microinjected. We used this method to perform single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in in mouse embryos. This method facilitates large-scale genetic analysis in the mouse.
Highlights
Recent use of the CRISPR/Cas[9] system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis
To achieve efficient genome editing in mouse embryos using electroporation, we started by optimizing the electroporation conditions for introducing mRNA into fertilized mouse eggs with an intact zona pellucida
We introduced Cas[9] mRNA and a gRNA targeting Fgf[10] (#563), which was previously transferred into eggs by the microinjection method and elicited the limbless phenotype[6] (Fig. 2a)
Summary
Recent use of the CRISPR/Cas[9] system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis. Among efficient genome editing methods, the CRISPR/Cas[9] system is the simplest for generating mice carrying a modified genome[2,3,4]. To achieve efficient genome editing in mouse embryos using electroporation, we started by optimizing the electroporation conditions for introducing mRNA into fertilized mouse eggs with an intact zona pellucida.
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