Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events

Qingyi Lin,Maki Hirata,Manita Wittayarat,Quynh Anh Le,Chommanart Thongkittidilok,Takeshige Otoi,Koki Takebayashi,Fuminori Tanihara

doi:10.1186/s13104-021-05800-8

Qingyi Lin, Maki Hirata + Show 6 more

Open Access

https://doi.org/10.1186/s13104-021-05800-8

Copy DOI

Abstract

ObjectiveLipofection-mediated introduction of the CRISPR/Cas9 system in porcine zygotes provides a simple method for gene editing, without requiring micromanipulation. However, the gene editing efficiency is inadequate. The aim of this study was to improve the lipofection-mediated gene editing efficiency by optimizing the timing and duration of lipofection.ResultsZona pellucida (ZP)-free zygotes collected at 5, 10, and 15 h from the start of in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA (gRNA) targeting GGTA1, and Cas9 for 5 h. Lipofection of zygotes collected at 10 and 15 h from the start of IVF yielded mutant blastocysts. Next, ZP-free zygotes collected at 10 h from the start of IVF were incubated with lipofection reagent, gRNA, and Cas9 for 2.5, 5, 10, or 20 h. The blastocyst formation rate of zygotes treated for 20 h was significantly lower (p < 0.05) than those of the other groups, and no mutant blastocysts were obtained. Moreover, the mutation rates of the resulting blastocysts decreased as the incubation time increased. In conclusion, a lipofection-mediated gene editing system using the CRISPR/Cas9 system in ZP-zygotes is feasible; however, further improvements in the gene editing efficiency are required.

Highlights

Pigs are excellent laboratory models for medical research owing to their similarity to humans, both anatomically and physiologically
Experiment 2 Zygotes collected at 10 h from the start of in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA (gRNA), and CRISPR-associated protein 9 (Cas9) for various durations
We have previously demonstrated that lipofection-mediated gene editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, without specialized equipment, can be performed in zona pellucida (ZP)-free oocytes and embryos [7]

Summary

Results

Experiment 1 The blastocyst formation rates did not differ significantly with respect to the timing of lipofection (Additional file 1: Table S1). Lipofection in zygotes collected at 10 and 15 h from the start of IVF resulted in the introduction of mosaic mutations in blastocysts. The mutation rates (Fig. 1a) and mutation efficiency (Fig. 1b) did not differ statistically between the two time points (11.1% vs 10.0% and 14.3% vs 13.0%, respectively). There were no mutant blastocysts derived from treated zygotes collected at 5 h from the start of IVF.`. Experiment 2 Zygotes collected at 10 h from the start of IVF were incubated with lipofection reagent, gRNA, and Cas for various durations. The blastocyst formation rate for zygotes treated for 20 h was significantly lower (p < 0.05) than those of the other groups, and no mutant blastocysts were obtained (Table 1).

Introduction

Main text

Discussion

Limitations