Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, 20892, USA.
Genomics Core, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, 20892, USA.
Sci Rep. 2019 Feb 7;9(1):1662. doi: 10.1038/s41598-018-33533-5.
A particular challenge in genome engineering has been the simultaneous introduction of mutations into linked (located on the same chromosome) loci. Although CRISPR/Cas9 has been widely used to mutate individual sites, its application in simultaneously targeting of linked loci is limited as multiple nearby double-stranded DNA breaks created by Cas9 routinely result in the deletion of sequences between the cleavage sites. Base editing is a newer form of genome editing that directly converts C∙G-to-T∙A, or A∙T-to-G∙C, base pairs without introducing double-stranded breaks, thus opening the possibility to generate linked mutations without disrupting the entire locus. Through the co-injection of two base editors and two sgRNAs into mouse zygotes, we introduced C∙G-to-T∙A transitions into two cytokine-sensing transcription factor binding sites separated by 9 kb. We determined that one enhancer activates the two flanking genes in mammary tissue during pregnancy and lactation. The ability to introduce linked mutations simultaneously in one step into the mammalian germline has implications for a wide range of applications, including the functional analysis of linked cis-elements creating disease models and correcting pathogenic mutations.
基因组工程的一个特殊挑战是同时将突变引入连锁（位于同一染色体上）基因座。虽然 CRISPR/Cas9 已被广泛用于突变单个位点，但由于 Cas9 产生的多个附近双链 DNA 断裂通常导致在切割位点之间删除序列，因此其在同时靶向连锁基因座方面的应用受到限制。碱基编辑是一种较新的基因组编辑形式，它可以直接将 C∙G 转换为 T∙A，或 A∙T 转换为 G∙C，而不会引入双链断裂，从而为不破坏整个基因座的连锁突变创造了可能性。通过将两个碱基编辑器和两个 sgRNA 共注射到小鼠受精卵中，我们在两个被 9kb 分隔的细胞因子感应转录因子结合位点中引入了 C∙G 到 T∙A 的转换。我们确定，一个增强子在怀孕和哺乳期激活乳腺组织中两个侧翼基因。在哺乳动物生殖系中一步同时引入连锁突变的能力具有广泛的应用意义，包括创建疾病模型和纠正致病突变的连锁顺式元件的功能分析。
