i- GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats

摘要

Zygote-microinjection or in vitro electroporation of isolated zygotes are now widely used methods to produce genome-edited mice. However, these technologies require laborious and time-consuming ex vivo handling of fertilized eggs, including zygote isolation, gene delivery into zygotes and embryo transfer into recipients. We recently developed an alternative method called improved genome-editing via oviductal nucleic acids delivery (i-GONAD), which does not require the above-mentioned ex vivo handing of zygotes, but instead involves intraoviductal instillation of genome-editing components, Cas9 protein and synthetic gRNAs, into the oviducts of pregnant females at the late 1-cell embryo stage under a dissecting microscope and subsequent electroporation. With this method, we succeeded in generating genome-edited mice at relatively high efficiencies (for example, knockout alleles were produced at ~97% efficiency). Here, we extended this improved technology to rats, and found that i-GONAD can create genome-edited rats in various strains, including Sprague Dawley and Lewis, and F1 hybrids (between Sprague Dawley and Brown Norway), with efficiencies of ~62% for indel mutations and ~9% for knock-ins. Thus, i-GONAD will be especially useful for the production of genome-edited rats in small laboratories where expensive micromanipulator systems and highly skilled personnel for embryo manipulation are unavailable.