i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases

Abstract

We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1400-x) contains supplementary material, which is available to authorized users.

i‐GONAD: A method for generating genome‐edited animals without ex vivo handling of embryos

“…These results are probably explained by the presence of cumulus cells surrounding the zygotes at Day 0.4; however, by Day 0.7, connections of these cumulus cells become loose, which allows eGFP mRNA solution to reach close to zygotes. Based on this experiment, we conclude that Day 0.7 is the optimal time for GONAD treatment (Ohtsuka et al., ).…”

“…Pregnant females on the day of having copulation plug contain one‐cell stage embryo in the ampulla (in which zygotes exist massively), the dilated area of the oviduct where the fertilization occurs. To assess the optimal time for GONAD, we injected eGFP mRNA into the oviductal lumen of pregnant females on Day 0.4 and Day 0.7, and performed in vivo EP (Ohtsuka et al., ). Observation of embryos from the two‐cell to blastocyst stage showed eGFP fluorescence only in the embryos derived from females subjected to GONAD on Day 0.7 of pregnancy.…”

Periodic patterns in Rodentia: Development and evolution

“…The advent of CRISPR/Cas9 technology has opened the door for performing a wide variety of genome editing experiments in both “traditional” and “non‐traditional” model systems, allowing a wide variety of modifications, including gene and enhancer knockouts, transcriptional activation and repression, and insertions of large genomic regions. Furthermore, recent studies in laboratory mice have achieved high genome editing efficiencies by delivering the CRISPR/Cas9 cocktail directly into zygotes inside the female oviduct, using electroporation or adeno‐associated viruses, thereby bypassing some of the hurdles associated with the traditional approach, such as the use of sophisticated micromanipulation equipment for ex vivo zygote microinjection and embryo transfer, usually performed by highly trained personnel. Incorporating these approaches in striped mice holds the promise of achieving precise and efficient genome modification to probe developmental mechanisms underlying pigment pattern formation.…”

The p21 dependent G2 arrest of the cell cycle in epithelial tubular cells links to the early stage of renal fibrosis

“…Mice C57BL/6 mice were used all in this study. p21 deficient mice were generated by i-GONAD method as previously described (34). In brief, the single strand DNA which contains 3 STOP codons and All animal experiments were approved by the institutional committee.…”

“…To investigate the impact of p21 in renal fibrosis, we constructed the novel p21 deficient mice (p21 -/-). To this end, we took the advantages of i-GONAD (improved-Genome-editing via oviductal nucleic acid delivery) method, in which handling the fertilized egg ex vivo is not required (33,34). Tandem STOP codons were integrated into 60 bases after the first ATG (Fig.…”