Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo, Japan.
Methods Mol Biol. 2023;2637:181-194. doi: 10.1007/978-1-0716-3016-7_14.
In the CRISPR/Cas9-mediated gene cassette knockin (KI) strategy, a gene cassette is integrated into a target locus through a proper DNA repair pathway after the Cas9-induced double-strand DNA breaks; the activation of the DNA repair pathway is known to be correlated with the cell cycle. Recently, we have reported a new KI approach named SPRINT (S-phase pronuclear injection for targeting)-CRISPR, focusing on the correlation between the cell cycle and the KI efficiency in the mouse zygote microinjection. Our results suggest that the CRISPR-mediated KI with a homologous recombination-based donor vector during S-phase enhances the KI efficiency. For SPRINT-CRISPR, the uniformity of the zygotes in the cell cycle is achieved by in vitro fertilization, and the zygotes are cryopreserved until use. These reproductive techniques are necessary for efficient KI. Furthermore, Piezo-assisted microinjection has been successful in improving the survival rate of the injected embryos. In this chapter, we describe the protocols that focus on the zygote preparation and Piezo-assisted microinjection of the SPRINT-CRISPR method.
在 CRISPR/Cas9 介导的基因盒敲入 (KI) 策略中，Cas9 诱导双链 DNA 断裂后，通过适当的 DNA 修复途径将基因盒整合到靶位点；已知 DNA 修复途径的激活与细胞周期相关。最近，我们报道了一种名为 SPRINT（靶向 S 期核内注射的 CRISPR）的新 KI 方法，重点研究了细胞周期与小鼠受精卵微注射中 KI 效率之间的相关性。我们的结果表明，在 S 期使用基于同源重组的供体载体进行 CRISPR 介导的 KI 可提高 KI 效率。对于 SPRINT-CRISPR，通过体外受精使受精卵在细胞周期中保持均匀，并将受精卵冷冻保存以备使用。这些生殖技术对于高效 KI 是必要的。此外，Piezo 辅助微注射已成功提高了注射胚胎的存活率。在本章中，我们描述了专注于 SPRINT-CRISPR 方法的受精卵准备和 Piezo 辅助微注射的方案。
