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.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, in which the Cas[9] 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
We found that injection of RNA into the cytoplasm yields the best developmental efficiency at the blastocyst stage and in producing full-term mouse pups (Figure 2)
This result suggests that the injection of DNA or RNA into the pronucleus is detrimental to the viability of the embryos
Summary
The CRISPR/Cas system, in which the Cas[9] 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. 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 showed the best overall knockout efficiency. We found that the www.nature.com/scientificreports injection of RNA into the cytoplasm was the most efficient method and yielded the greatest numbers of normal blastocyst stage embryos and full-term pups.
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