Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice

Abstract

Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation, in vitro fertilization (IVF) and vitrification/warming of zygotes. We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice.

Electroporation-mediated genome editing in vitrified/warmed mouse zygotes created by IVF via ultra-superovulation

“…Subsequently, the gRNAs were synthesized using a MEGAshortscript T7 Kit (Thermo Fisher Scientific, Tokyo, Japan), and then purified with a MEGAclear Kit (Thermo Fisher Scientific). The gRNA designs of interleukin-11 (Il11), tyrosinase ( Tyr ), and secreted phosphoprotein 1 ( Spp1 ) genes were as described previously [ 20 , 21 ]. In the generation of Il11 mutant mice, gRNA B was synthesized and used as shown in a previous report.…”

“…Cas9 Nuclease 3NLS; Tokyo, Japan). The sequence of ssODN for three-base substitution at the Spp1 locus was the same as in previous reports [ 20 , 21 ]. ssODN was synthesized by Integrated DNA Technologies (Coralville, IA, USA).…”

“…To achieve flexible scheduling and beneficial work efficiency, we previously reported the generation of various genome-edited mice by microinjection into vitrified/warmed zygotes. Efficient production of GM mice was confirmed by combining genome-editing technology with reproductive engineering techniques [ 17 , 19 , 20 , 21 ], such as our IVF method, providing high fertility using methyl-beta-cyclodextrin (MBCD)-treated sperm and reduced glutathione (GSH)-treated oocytes (CARD method) [ 26 ]; ultra-superovulation treatment of female mice, by which approximately 2–3 times as many oocytes can be collected per female mouse compared with the conventional superovulation method [ 27 ]; and vitrification/warming of fertilized oocytes [ 16 ]. However, the applicability of vitrified/warmed zygotes created by such advanced reproductive engineering technologies in electroporation-mediated genome editing has not yet been fully investigated.…”

“…Fresh zygotes were used for electroporation after 6.5–7.5 h from insemination (around E0.3). Vitrified/warmed zygotes were cultured for 1–5 h and then used for electroporation at different timings (E0.3–E0.5), similar to the approach using microinjection as described in our previous report [ 21 ]. After electroporation, the surviving one-cell zygotes or two-cell embryos were transferred to pseudopregnant female mice after culture for about 1 h or overnight, to examine whether different birth rates occurred.…”

Magic wands of CRISPR—lots of choices for gene knock-in

“…In addition, the detailed protocol, freely available materials, and online design tool for the CRISPR-mediated PITCh knock-in were provided by the authors Nakamae et al 2017). Along with these user-friendly resources, the application examples of CRISPR-mediated PITCh have become more diversified, e.g., high-throughput epitope tagging in cultured cells for chromatin immunoprecipitation (ChIP)-seq (Xiong et al 2017), the application in mammalian zygotes (Aida et al 2016;Nakagawa et al 2017), and in vivo somatic knock-in in mice (Yao et al 2017a) were reported one after another. Note that recent design of CRISPR-mediated PITCh has employed longer microhomologies (~40 bp); thus, it might be based on SSA rather than MMEJ.…”