Generation of CRISPR-Edited Rodents Using a Piezo-Driven Zygote Injection Technique.

Direct modification of the genome of the zygotes (i.e., one-cell embryos) by the CRISPR/Cas9-editing reagents, followed by embryo transfer to pseudopregnant females for live birth, has been the most effective method to generate laboratory rodent models for research. The method relies on proper delivery of the editing reagents into zygotes, which is commonly achieved by a standard or slightly modified pronuclear microinjection technique. In this chapter, we describe in detail an alternative delivery method, named piezo-driven cytoplasmic microinjection, which offers a superior embryo survival and birth rate. Because this method uses a much wider injection needle than that in pronuclear injection, it allows a larger volume of the editing materials to be transported into the zygotes, leading to an increase in the targeting efficiency. This also eliminates the clogging issues seen regularly in pronuclear injection. Moreover, Cytochalasin B that is used to soften zygotes during piezo-driven microinjection has been suggested a role in improving the knockin efficiency, which provides an additional benefit to use this injection method.