Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice

Abstract

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.

Mouse Genome Editing from Scratch

“…We observed an unusually high level of cannibalism among test females so that about 75% of pups did not survive the first days of life, which agrees with the data published previously (Carter et al ., 2002). While this report was prepared, Melo-Silva and co-authors published their results on adaptation of the i-GONAD method in C57BL/6, where they describe similar challenges (Melo-Silva et al ., 2023). The authors suggest co-housing synchronized pregnant C57BL/6 mice with companion pregnant FVB/NJ females, so that the later assist in pup survival (Melo-Silva et al ., 2023).…”

“…While this report was prepared, Melo-Silva and co-authors published their results on adaptation of the i-GONAD method in C57BL/6, where they describe similar challenges (Melo-Silva et al ., 2023). The authors suggest co-housing synchronized pregnant C57BL/6 mice with companion pregnant FVB/NJ females, so that the later assist in pup survival (Melo-Silva et al ., 2023). This could be a great addition to the presented here setup as it might significantly improve i-GONAD efficiency.…”

“…We use superovulation in C57BL/6 female mice, since it was shown previously that genetic modification in inbred mouse strains of interest such as C57BL/6 is still challenging because of their low fertility and embryo survival. (Melo-Silva et al ., 2023) The i-GONAD procedure should be carried out on day 0.7 after fertilization (d.p.c., days past coating) (Ohtsuka et al ., 2018), thus we used the following protocol for mating females with males adapted to the light regime of our animal facility (12 h/12 h light/dark photoperiod, [00:00- 12:00-00:00]):…”