Summary
Recently, the CRISPR/Cas9 system has been widely used in the construction of mutant mice, but the use of microinjection methods has largely limited the application of gene editing to high throughput. In this article, we describe a simple, efficient, and large-scale method of gene editing: transferring RNAs into eggs by means of electroporation, rather than by microinjection. We used this method to transfer single-stranded oligodeoxynucleotides (ssODN) into mouse eggs to construct knockout mutants. The results of the study prove that this method makes large-scale genetic analysis a reality.
Materials and Method
Mouse, egg and embryo
Pregnant rats were used with ICR and B6D2F1 (C57BL/6XDBA2F1). ICR is mainly used for electrical rotation conditions, while B6D2F1 is used for gene editing.
The fertilized eggs were collected from the vas deferens of E0.5. ICR and B6D2F1 naturally hybridize with the same male. In the gene editing assay, eggs obtained from B6D2F 1 and R26-H2b-mCherry male crosses were cultured in mWM medium for electrotransfection.
Electrical transfection
Application BEX custom electrode (length: 10mm, width: 3mm, height: 0.5mm, spacing: 1mm) Figure 1.
The electrodes were attached to the CUY21EDIT II main unit (BEX, Tokyo, Japan) and the electrodes were placed under a stereo microscope. Eggs cultured in mWM were washed three times with Opti-MEM I. 5 ul of RNA was added for mixing and placed in the electrode slit. Can rotate 40-50 embryos. The electrical rotation condition is set to 30V, 7times. Immediately after electroporation, the eggs were transferred out of mWM medium and incubated at 37 ° C, 5% CO 2 . mCherry fluorescence was observed 15 h after transfection.
results and analysis
Transfection with mCherry mRNA and the limbless gene Fgf10, respectively, yielded a 94-95% ratio of viable fertilized eggs that survived and developed into two cell stages, much higher than the microinjection method (34-35%).
When we transfected with 400 ng/ul of Cas9 mRNA and 200 ng/ul gRNA, 87% of the embryos showed anterior and posterior legs, as shown in Figure 2c (type I); the type I mutant was sequenced and the Fgf10 gene was found. Alleles were all interrupted.
Subsequently, we examined the efficiency of gene editing for different concentrations of Cas9 mRNA and gRNA. 200 ng/ul Cas9 mRNA and 50 ng/u gRNA, 73% embryos without limbs; 100 ng/ul Cas9 mRNA and 50 ng/ul gRNA, 32% showing limb defects (Fig. 2bc); 50 ng/ul Cas9 mRNA and 25 ng/ul The gRNA, the embryo is mostly normal.
To further determine the efficiency of genome editing, mCherry mRNA was transferred into 11 mice and all 11 mice were found to be free of red fluorescence (Fig. 3b). This result suggests that the method of electroporation can also be used to efficiently generate HDR-mediated knockout mice.
Next, we explored whether this mutation will be inherited in the next generation. 200 ng/ul Cas9 mRNA and 100 ng/ul gRNA were electroporated into egg cells to target the mCherry gene. Twenty-five F0 mice were observed, and no red fluorescence was expressed. Moreover, all F1 mice did not have red fluorescence, indicating that the mCherry gene was interrupted. The above discloses that the genetic editing mutant obtained by the electroporation method can be stably inherited.
Figure 2. CRISPR/Cas9 gene editing by electroporation. Figure 3. Deletion mutants formed by electroporation of mCherry RNA.
discuss
In summary, we have established a highly efficient and high survival CRISPR/Cas9 gene editing method by means of electroporation. Because the method of electroporation can process 40-50 embryos simultaneously without the need of microinjection technology, it is simpler and faster to construct mouse mutants. It is convenient for large-scale rapid analysis of F0 phenotypes and other research.
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