Department of Biochemistry, University of Colorado at Boulder, Boulder, CO, 80309, USA.
Department of Genetics, University of Georgia, Athens, GA, USA.
Nat Commun. 2020 Mar 13;11(1):1394. doi: 10.1038/s41467-020-15226-8.
CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on regulation of CRISPR interference or editing in mammalian cells, none of the reported methods enable control of the nuclease activity in bacteria. Here, we develop RNA linkers to combine theophylline- and 3-methylxanthine (3MX)-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Escherichia coli. These activatable guide RNAs enable temporal and post-transcriptional control of in vivo gene editing. Further, they reduce the death of host cells caused by cuts in the genome, a major limitation of CRISPR-mediated bacterial recombineering.
CRISPR-Cas9 技术在广泛的应用领域中推动了基因编辑的重大进展。为了进一步提高 Cas9 的实用性，人们一直在努力实现对其核酸酶活性的时间控制。虽然不同的方法侧重于调节哺乳动物细胞中的 CRISPR 干扰或编辑，但没有一种报道的方法能够控制细菌中的核酸酶活性。在这里，我们开发了 RNA 接头，将茶碱和 3-甲基黄嘌呤（3MX）结合适体与 sgRNA 结合，使小分子依赖性编辑能够在大肠杆菌中进行。这些可激活的向导 RNA 能够实现体内基因编辑的时间和转录后控制。此外，它们减少了由基因组切割引起的宿主细胞死亡，这是 CRISPR 介导的细菌重组的主要限制。
