BRC Current Technology August 2021

RIKEN BRC BRC Current Technology
August 2021

17. Highly efficient generation of
KO founder mice with CRISPR/Cas9

Simultaneous disruption of multiple genes

Figure 1. An approach to generate knockout (KO) founder mice by CRISPR.
(a) Design guide RNAs for two or more different locations of protein-coding exons of a target gene.
(b) When the guide RNA designed as described above is injected into fertilized eggs together with Cas9 mRNA, the gene function can be disrupted with very high efficiency (95-100%).

Figure 2. Abnormalities in germ cell development caused by simultaneous disruption of the Tuba3a and Tuba3b genes.
Founder KO mice of single Tuba3a or Tuba3b gene showed no particular abnormalities, but double KO founder mice showed marked testicular atrophy and complete loss of DDX4-positive germ cells.

The phenotypic analysis of knockout (KO) mice has been performed through 1) generation of founder mice, 2) expansion of colony by breeding, and then 3) generation of knockout mice through crossbreeding between heterozygous mice. Such an approach required several generations of crosses, making the analysis very time-consuming. The CRISPR/Cas9 technology allows genome editing with very high efficiency. It has been reported that biallelic KO founder mice can be generated with high efficiency by simultaneously injecting multiple guide RNAs targeting a single gene together with Cas9 mRNA [1]. We have succeeded in generating KO founder mice with nearly 100% efficiency using this approach [2]. We used this approach to disrupt two genes at the same time to directly generate double KO founder mice [3]. We found that simultaneous disruption of the Tuba3a and Tuba3b genes resulted in complete loss of testicular germ cells. Such simultaneous disruption of multiple genes will be very useful as a phenotypic screening method.


[1] E. A. Susaki et al., “Next-generation mammalian genetics toward organism-level systems biology,”
npj Sys. Biol. Appl., 3, 1–10, 2017.
[2] S. Matoba et al., “Paternal knockout of Slc38a4/SNAT4 causes placental hypoplasia associated with intrauterine growth restriction in mice,”
Proc. Natl. Acad. Sci. U. S. A., 116, 21047–21053, 2019.
[3] M. S. Akter et al., “CRISPR/Cas9-based genetic screen of SCNT-reprogramming resistant genes identifies critical genes for male germ cell development in mice,”
Sci. Rep., DOI: 10.1038/s41598-021-94851-9, 2021.

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