Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing

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.

Figure 1. Optimal conditions for the electroporation used to deliver mRNA into fertilized eggs.

(a) Electroporation set-up used in this study. The right box is the electroporator CUY21 EDIT II (BEX Co. Ltd.), which generates electric pulses, and the left is a stereoscopic microscope for embryo manipulation. Two electrodes were placed on the microscope stage and connected to the electroporator. (b) Higher magnification of the red rectangle in (a). (c) Schematic of the electroporation chamber with customized platinum electrodes (BEX Co. Ltd.). Fertilized mouse eggs were placed in the RNA solution in the gap between the electrodes. (d) Microscopic view of the eggs set in the electrode gap. The eggs were manually positioned into a line prior to electroporation. (e) Schematic of the electroporation conditions used to introduce RNAs into mouse eggs: three to eleven repeats of a square pulse of 10–50 V; 3-msec pulses with 97-msec intervals were used. (f) Fluorescence intensity of mCherry (red circles) and rate of electroporated embryo survival to the blastocyst stage (blue squares) were plotted at various voltages (n = 10). The duration of each pulse and number of pulse repeats were fixed at 3 msec and 5 repeats, respectively. (g) The fluorescent intensity of mCherry (red closed circle) and the survival rate of the electroporated embryos to the blastocyst stage (blue squares) were plotted as a function of the number of electroporation repeats (n = 12). The voltage and duration of each pulse were fixed at 30 V and 3 msec, respectively.

Figure 2. CRISPR/Cas9-mediated genome editing by electroporation.

(a) Genomic structure of the Fgf10 locus, which includes the target sequence (underlined) and the protospacer-adjacent motif (PAM) sequence (shown in blue), used in this study. (b) Examples of embryos from the three classes that exhibited different limb phenotypes: no limbs (type I), limb defects (type II, left: hindlimb deficiency, right: truncated fore- and hind-limb), and normal (type III). (c) Graph summarizing the effects of Cas9 and gRNA electroporation on limb development. The concentrations of RNAs used in each experiment are shown at left. The numbers in each column are the number of embryos exhibiting the phenotype in each category.

Figure 3. The single-stranded oligodeoxynucleotide (ssODN)-induced generation of HDR-mediated insertions.

(a) Schematic of the target sequence and the ssODN designed to insert the 37-base loxP sequence (shown in green) and EcoRI recognition site (shown in red). (b) Representative embryo electroporated with Cas9 mRNA, gRNA, and ssODN. The mCherry fluorescence completely disappeared in the electroporated embryos, while control (no electroporation) embryos displayed fluorescent signals. (c) RFLP analysis of the collected embryos. The EcoRI-inserted alleles were digested into two bands (138 bps and 374 bps). The intact allele had 497 bps. The digested bands were observed in embryos # 3, 6, 8, and 9. The unexpected bands in #1 and #7 were the target sequence containing a large deletion.