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. 2014 Mar 28;4:4513.
doi: 10.1038/srep04513.

Validation of Microinjection Methods for Generating Knockout Mice by CRISPR/Cas-mediated Genome Engineering

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Free PMC article

Validation of Microinjection Methods for Generating Knockout Mice by CRISPR/Cas-mediated Genome Engineering

Takuro Horii et al. Sci Rep. .
Free PMC article

Abstract

The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. The Cas9/gRNA-targeting site in mouse Tet1 and validation of its targeting efficiency.
(a) The Cas9/gRNA-targeting sites in mouse Tet1. The gRNA-targeting sequence is underlined and the PAM sequences are indicated in red. Exons are indicated by closed boxes and the boxed sequence indicates the Sac I restriction site in the target region. (b) Validation of targeting efficiency of the Tet1 gene using mouse embryonic stem cells. PCR products were digested with the restriction enzyme Sac I that cleaves at the Cas9 endonuclease target site and then analyzed by gel electrophoresis. PCR products generated from DNA containing successfully targeted Tet1 were uncleaved and, therefore, larger than the product generated from the control DNA. The intensity of each fragment was measured and the targeting efficiency was calculated.
Figure 2
Figure 2. Viability of the Tet1 targeted mice.
(a) Percentage of viable blastocysts per injected embryos. *, P < 0.05. (b) Percentage of pups per transferred embryos. *, P < 0.05.
Figure 3
Figure 3. Newborn mice generated by the CRISPR method.
Five pups generated by each injection method are shown. Tet1 genotypes are indicated in each image.
Figure 4
Figure 4. Knockout efficiencies of each injection method.
(a) Percent of each Tet1 genotype among all newborn mice generated by each injection method. (b) Overall efficiencies of generating knockout mice (+/− or −/−) by each of the three injection methods. Numbers of knockout mice per transferred embryos are presented. *, P < 0.05.
Figure 5
Figure 5. Analysis of mutations generated by CRISPR.
Genotyping of newborn mice generated by the CRISPR method. The two Tet1 alleles are presented for each mouse. The PAM sequences are shown in red. Deletions are indicated in grey letters. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions. Micro-homology flanking the cleavage site is underlined in the sequence.

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