背景:昼夜和夜间哺乳动物已经进化出不同的途径来优化其特定于时间型的生活方式的生存。传统的啮齿动物模型,夜间活动,可能不足以概括人类在健康和疾病方面的昼夜生物学。尽管昼夜啮齿动物可能有利于转化研究,直到最近,他们没有基因处理。本研究旨在通过在建立良好的昼夜啮齿动物模型中开发基因组编辑所需的实验程序来解决这一主要限制。尼罗河草鼠(Arvicanthisniloticus)。
结果:建立了超排卵方案,每只雌性草鼠产卵近30个。受精卵在改良的大鼠1细胞胚胎培养基(mR1ECM)中培养,其中草鼠胚胎从1细胞阶段发育成胚泡。然后将基于CRISPR的方法用于体内和体外的基因编辑,靶向维甲酸诱导的1(Rai1),Smith-Magenis综合征的致病基因,神经发育障碍.使用经由输卵管核酸递送(i-GONAD)方法的改进的基因组编辑,通过电穿孔在体内递送CRISPR试剂。体内方法产生了几只编辑过的具有Rai1无效突变的创始人草大鼠,这表明目标等位基因稳定地传递给下一代。CRISPR试剂也在体外显微注射到2细胞胚胎中。在70%的胚胎中证实了Rai1基因的大缺失,展示高效的体外基因组编辑。
结论:我们已经建立了一套方法,能够在尼罗河草大鼠中首次成功进行基于CRISPR的基因组编辑。开发的方法将指导未来对这种和其他昼夜啮齿动物物种的基因组编辑,这将促进这些模型在基础和转化研究中的更大效用。
BACKGROUND: Diurnal and nocturnal mammals have evolved distinct pathways to optimize survival for their chronotype-specific lifestyles. Conventional rodent models, being nocturnal, may not sufficiently recapitulate the biology of diurnal humans in health and disease. Although diurnal rodents are potentially advantageous for translational research, until recently, they have not been genetically tractable. The present study aims to address this major limitation by developing experimental procedures necessary for genome editing in a well-established diurnal rodent model, the Nile grass rat (Arvicanthis niloticus).
RESULTS: A superovulation protocol was established, which yielded nearly 30 eggs per female grass rat. Fertilized eggs were cultured in a modified rat 1-cell embryo culture medium (mR1ECM), in which grass rat embryos developed from the 1-cell stage into blastocysts. A CRISPR-based approach was then used for gene editing in vivo and in vitro, targeting Retinoic acid-induced 1 (Rai1), the causal gene for Smith-Magenis Syndrome, a neurodevelopmental disorder. The CRISPR reagents were delivered in vivo by electroporation using an improved Genome-editing via Oviductal Nucleic Acids Delivery (i-GONAD) method. The in vivo approach produced several edited founder grass rats with Rai1 null mutations, which showed stable transmission of the targeted allele to the next generation. CRISPR reagents were also microinjected into 2-cell embryos in vitro. Large deletion of the Rai1 gene was confirmed in 70% of the embryos injected, demonstrating high-efficiency genome editing in vitro.
CONCLUSIONS: We have established a set of methods that enabled the first successful CRISPR-based genome editing in Nile grass rats. The methods developed will guide future genome editing of this and other diurnal rodent species, which will promote greater utility of these models in basic and translational research.