使用三种非病毒载体转染试剂:非脂质体聚合物(TransIT-X2)比较了成簇的规则间隔短回文重复序列(CRISPR)/Cas核糖核蛋白复合物的转染效率,脂质纳米颗粒递送(CRISPRMAX),和肽(ProteoCarry)系统。将猪无透明带的受精卵和胚胎与CRISPR相关蛋白9(Cas9)孵育5小时,引导RNA(gRNA)靶向的GGTA1,和试剂之一。在实验1中,优化Cas9蛋白与gRNA的摩尔比为1:2、2:2和4:2,以及单剂量或双剂量的试剂,在体外受精后10小时对受精卵进行。在实验2中,在体外受精后10或29小时进行了时间优化,使用最佳摩尔比和试剂剂量。囊胚形成,突变率,并在每个实验中测量突变效率。对于每种试剂,a4:2Cas9:gRNA摩尔比和添加双倍试剂剂量显示出较高的突变率;然而,与对照组相比,囊胚率趋于下降。此外,最佳转染时间因试剂而异,携带突变的胚泡比例<34%。总之,上述三种转染子允许对猪受精卵和胚胎进行基因编辑;然而,这项新建立的基于化学的技术需要进一步改进,特别是关于编辑效率和胚胎发育。
The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5 h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10 h post-in vitro fertilization. In Experiment 2, optimization of timing was performed at 10 or 29 h post-in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were <34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry-based technology needs further improvement, especially regarding editing efficiency and embryo development.