A CRISPR/Cas-Based Method for Precise DNA Integration in Xenopus laevis Oocytes Followed by Intracytoplasmic Sperm Injection (ICSI) Fertilization.
A CRISPR/Cas-Based Method for Precise DNA Integration in Xenopus laevis Oocytes Followed by Intracytoplasmic Sperm Injection (ICSI) Fertilization.
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基于 CRISPR/Cas 的非洲爪蟾卵母细胞精确 DNA 整合方法，随后进行胞浆内单精子注射 (ICSI) 受精。
DOI：
10.1007/978-1-0716-3004-4_11
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Martin SA
中科院分区：
文献类型：
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作者：
Martin SA
非洲爪蟾长期以来因其作为用于发育细胞和生物化学研究的强大模式生物而享有盛誉。随着基因编辑技术的出现，以及非洲爪蟾基因组的全基因组测序揭示了非洲爪蟾与人类之间的遗传保守程度，非洲爪蟾有潜力成为人类遗传疾病的理想模型。然而，无法通过同源定向修复产生非镶嵌、精确的 DNA 插入，限制了非洲爪蟾这一领域的实力。此外，它还阻止研究人员充分利用融合标签，这是一种用表位或荧光标签直接标记基因的方法，允许在不使用蛋白质特异性抗体的情况下对蛋白质进行可视化、定量和跟踪。在这里，我们描述了一种使用 CRISPR/Cas9 将 DNA 精确插入卵母细胞的方法，然后通过胞浆内单精子注射 (ICSI) 进行体外成熟和受精，最终产生携带非马赛克杂合插入的胚胎。
Xenopushas long had a reputation for being a powerful model organism for use in developmental cell and biochemistry research. With the advent of gene-editing technologies, and the full genome sequencing ofXenopusgenomes revealing the extent of the genetic conservation betweenXenopusand humans,Xenopushas the potential to become an ideal model for human genetic disease. However, the inability to produce non-mosaic, precise DNA insertions through homology directed repair has limited the strength ofXenopusthis field. Furthermore, it has prevented researchers from taking full advantage of fusion tagging, a method for directly tagging genes with either epitope or fluorescent tags, allowing the visualization, quantification, and tracking of proteins without the use of protein-specific antibodies. Here, we describe a method for precise DNA insertion into oocytes using CRISPR/Cas9, followed by in vitro maturation and fertilization by intracytoplasmic sperm injection (ICSI), culminating in the production of embryos carrying a non-mosaic, heterozygous insertion.