Lin Jason C, Van Eenennaam Alison L
Department of Animal Science, University of California, Davis, Davis, CA, United States.
Front Genet. 2021 Apr 13;12:648482. doi: 10.3389/fgene.2021.648482. eCollection 2021.
The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.
传统上，将基因组编辑试剂导入哺乳动物受精卵是通过细胞质或原核显微注射来完成的。这个耗时的过程需要昂贵的设备和高水平的技术。受精卵电穿孔为转染哺乳动物受精卵提供了一种更简化、更高效的方法。有许多研究探讨了小鼠和大鼠受精卵电穿孔所使用的参数。在这里，我们除了回顾已报道的猪和牛等家畜受精卵的少数研究外，还综述了小鼠和大鼠的电穿孔条件、时间安排和成功率。在受精时或受精后不久引入编辑试剂有助于降低嵌合体的比例，即个体细胞中存在两种或更多种基因型的情况；引入核酸酶蛋白而非编码核酸酶的mRNA也有助于降低嵌合体比例。嵌合体在世代间隔长的大型家畜物种中尤其成问题，因为通过育种获得非嵌合的纯合后代可能需要数年时间。与基因敲入相比，通过非同源末端连接途径实现的基因敲除已有更广泛的报道，并且使用电穿孔已成功完成。透明带（ZP）阻碍了将大型DNA质粒导入受精卵，通过电穿孔完成的大多数基因敲入使用的是短单链DNA（ssDNA）修复模板，通常小于1 kb。在不使用细胞质注射的情况下，将长达4.9 kb的更大供体修复模板与基因组编辑试剂一起导入受精卵的最有前景的方法是将重组腺相关病毒（rAAV）与电穿孔结合使用。然而，与用于递送成簇规律间隔短回文重复序列（CRISPR）基因组编辑试剂的其他方法类似，这种方法也与高水平的嵌合体有关。最近用经过编辑的具有生殖系能力的细胞补充生殖系消融个体的进展提供了一种避免基因组编辑建系动物生殖系中出现嵌合体的方法。即使通过电穿孔介导将基因组编辑试剂递送至哺乳动物受精卵，基因组编辑流程中仍存在其他瓶颈，目前这些瓶颈阻碍了非嵌合基因组编辑家畜的规模化生产。
