A versatile toolbox for knock-in gene targeting based on the Multisite Gateway technology

Knock-in (KI) gene targeting can be employed for a wide range of applications in stem cell research. However, vectors for KI require multiple complicated processes for construction, including multiple times of digestion/ligation steps and extensive restriction mapping, which has imposed limitations for the robust applicability of KI gene targeting. To circumvent this issue, here we introduce versatile and systematic methods for generating KI vectors by molecular cloning. In this approach, we employed the Multisite Gateway technology, an efficient in vitro DNA recombination system using proprietary sequences and enzymes. KI vector construction exploiting these methods requires only efficient steps, such as PCR and recombination, enabling robust KI gene targeting. We show that combinatorial usage of the KI vectors generated using this method and site-specific nucleases enabled the precise integration of fluorescent protein genes in multiple loci of human and common marmoset (marmoset; Callithrix jacchus) pluripotent stem cells. The methods described here will facilitate the usage of KI technology and ultimately help to accelerate stem cell research.

基因敲入（Knock-in, KI）基因靶向技术可应用于干细胞研究的诸多场景。然而，KI载体的构建需历经多步复杂流程，包括多次酶切/连接操作与大量限制性酶切图谱分析，这限制了KI基因靶向技术的稳定应用。为解决这一问题，本文介绍了基于分子克隆技术构建KI载体的通用化、系统化方法。该方法采用多位点Gateway技术——一种依托专有序列与酶的高效体外DNA重组系统。利用该方法构建KI载体仅需PCR扩增与重组反应等高效步骤，即可实现稳定可靠的KI基因靶向操作。研究证实，将本方法制备的KI载体与位点特异性核酸酶联合使用，可在人类及普通狨猴（狨猴；Callithrix jacchus）多能干细胞的多个基因组位点精准整合荧光蛋白基因。本文所述方法将有力推动KI技术的应用，最终助力干细胞研究的加速发展。