Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea.
Tissue Eng Regen Med. 2020 Feb;17(1):45-53. doi: 10.1007/s13770-019-00225-8. Epub 2019 Nov 19.
CRISPR/Cpf1 is a class II, type V RNA-guided endonuclease that is distinct from the type II CRISPR/Cas9 nuclease, widely used for genome editing. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR/Cas9 system. The applications of CRISPR to rodent embryos for the production of knock-out (KO) mice have been achieved mainly by microinjection, which requires heavily-equipped instruments with skillful hands. Here, we evaluated the genome editing efficiency between Cpf1/mRNA and Cpf1/ribonuclear protein (RNP) in mouse embryos, and established an easy, fast, and technically less demanding method to produce KO mice using electroporation of the Cfp1/RNP system.
The efficiency of electroporation-based delivery of AsCpf1/mRNA and AsCpf1/RNP to target exon 3 of leukemia inhibitory factor (Lif) into mouse zygotes was evaluated. Embryos that developed to the two-cell stage after zygote electroporation were transferred into the oviducts of surrogate mothers to produce AsCpf1-mediated LIF KO mice. The genome editing efficiency of blastocysts and pups was tested using the T7E1 assay and/or DNA sequencing. Congenital abnormalities and reproductive phenotypes in LIF KO mice produced by electroporation with AsCpf1/RNP were examined.
Survival and two-cell development of electroporated zygotes were comparable between the AsCpf1/mRNA and AsCpf1/RNP groups, whereas genome editing efficiency was relatively higher in the AsCpf1/RNP group (13.3% vs 18.1% at blastocyst and 33.3% vs 45.5% at offspring), respectively. Two mouse lines with a frameshift mutation in exon 3 of the Lif gene were established from the AsCpf1/RNP group. All congenital abnormalities of LIF KO mice produced by AsCpf1/RNP electroporation were observed. AsCpf1-mediated LIF KO mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of LIF KO mice generated by conventional gene targeting.
Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.
CRISPR/Cpf1 是一种不同于广泛用于基因组编辑的 II 类、V 型 RNA 指导的内切酶。Cpf1 是一种比 Cas9 更小、更简单的内切酶，克服了 CRISPR/Cas9 系统的一些限制。CRISPR 被应用于产生基因敲除（KO）小鼠的啮齿动物胚胎，主要通过微注射来实现，这需要使用配备精良的仪器和熟练的技术。在这里，我们评估了 Cpf1/mRNA 和 Cpf1/核糖核蛋白（RNP）在小鼠胚胎中的基因组编辑效率，并建立了一种使用 Cfp1/RNP 系统电穿孔产生 KO 小鼠的简单、快速且技术要求较低的方法。
评估了将 AsCpf1/mRNA 和 AsCpf1/RNP 电穿孔递送至白血病抑制因子（Lif）靶exon3 的效率，将电穿孔后的合子发育到二细胞期后移植到代孕母鼠的输卵管中以产生 AsCpf1 介导的 LIF KO 小鼠。使用 T7E1 测定法和/或 DNA 测序测试了囊胚和幼崽的基因组编辑效率。检查了用电穿孔 AsCpf1/RNP 产生的 LIF KO 小鼠的先天性异常和生殖表型。
AsCpf1/mRNA 和 AsCpf1/RNP 组的电穿孔合子的存活率和二细胞发育无差异，而 AsCpf1/RNP 组的基因组编辑效率相对较高（囊胚分别为 13.3%和 18.1%，幼崽分别为 33.3%和 45.5%）。从 AsCpf1/RNP 组中建立了两条 Lif 基因exon3 发生移码突变的小鼠品系。用电穿孔 AsCpf1/RNP 产生的所有 LIF KO 小鼠的先天性异常均被观察到。AsCpf1 介导的 LIF KO 小鼠表现出出生后生长迟缓和植入失败，这是通过传统基因靶向产生的 LIF KO 小鼠的主要表型。
在合子阶段电穿孔 AsCpf1/RNP 是一种产生 KO 小鼠的有效基因组编辑方法。
