Acosta Sandra, Fiore Luciano, Carota Isabel Anna, Oliver Guillermo
Center for Vascular & Developmental Biology, Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois, 60611.
Genesis. 2018 May;56(5):e23212. doi: 10.1002/dvg.23212. Epub 2018 May 11.
Targeted genome editing in mouse embryonic stem cells (ESCs) is a powerful resource to functionally characterize genes and regulatory elements. The use of the CRISPR/Cas9 genome editing approach has remarkably improved the time and efficiency of targeted recombination. However, the efficiency of this protocol is still far from ideal when aiming for bi-allelic homologous recombination, requiring at least two independent targeting recombination events. Here we describe an improved protocol that uses two gRNAs flanking the selected targeted region, leading to highly efficient homologous recombination in mouse ESCs. The bi-allelic recombination targeting efficiency is over 90% when using two gRNAs together with the inhibition of non-homologous end-joint repair. Moreover, this technique is compatible with the generation of knocked-in mice and the use of ESC-derived differentiation protocols, therefore facilitating and accelerating the gene targeting in mice and ESCs.
在小鼠胚胎干细胞（ESC）中进行靶向基因组编辑是从功能上表征基因和调控元件的强大资源。CRISPR/Cas9基因组编辑方法的使用显著提高了靶向重组的时间和效率。然而，当目标是双等位基因同源重组时，该方案的效率仍远不理想，这需要至少两个独立的靶向重组事件。在这里，我们描述了一种改进的方案，该方案使用位于选定靶向区域两侧的两个引导RNA（gRNA），从而在小鼠胚胎干细胞中实现高效同源重组。当使用两个gRNA并抑制非同源末端连接修复时，双等位基因重组靶向效率超过90%。此外，该技术与敲入小鼠的产生以及ESC衍生分化方案的使用兼容，因此便于并加速了小鼠和ESC中的基因靶向。
