Kumar Namrata, Stanford William, de Solis Christopher, Abraham Nigel D, Dao Trieu-Mi J, Thaseen Sadiqa, Sairavi Anusha, Gonzalez Cuauhtemoc Ulises, Ploski Jonathan E
School of Behavioral and Brain Sciences, Department of Molecular & Cell Biology, The University of Texas at Dallas, Richardson, TX, United States.
Front Mol Neurosci. 2018 Nov 13;11:413. doi: 10.3389/fnmol.2018.00413. eCollection 2018.
The RNA-guided Cas9 nuclease, from the type II prokaryotic clustered regularly interspersed short palindromic repeats (CRISPR) adaptive immune system, has been adapted by scientists to enable site specific genome editing of eukaryotic cells both and . Previously, we reported the development of an adeno-associated virus (AAV)-mediated CRISPR (Sp) Cas9 system, in which the genome editing function can be regulated by controlling the expression of the guide RNA (sgRNA) in a doxycycline (Dox)-dependent manner. Here, we report the development of an AAV vector tool kit utilizing the Cas9 from (SaCas9). We demonstrate genome editing in human derived 293FT cells and mouse derived Neuro2A (N2A) cells and in neurons of the mouse brain. We also demonstrate the ability to regulate the induction of genome editing temporally with Dox and spatially with Cre-recombinase. The combination of these systems enables AAV-mediated CRISPR/Cas9 genome editing to be regulated both spatially and temporally.
来自II型原核生物成簇规律间隔短回文重复序列（CRISPR）适应性免疫系统的RNA引导的Cas9核酸酶，已被科学家改造用于对真核细胞进行定点基因组编辑。此前，我们报道了腺相关病毒（AAV）介导的CRISPR（Sp）Cas9系统的开发，其中基因组编辑功能可通过以强力霉素（Dox）依赖的方式控制引导RNA（sgRNA）的表达来调节。在此，我们报道了利用来自（SaCas9）的Cas9开发的AAV载体工具包。我们在人源293FT细胞和鼠源Neuro2A（N2A）细胞以及小鼠大脑神经元中证明了基因组编辑。我们还展示了用Dox在时间上和用Cre重组酶在空间上调节基因组编辑诱导的能力。这些系统的组合使AAV介导的CRISPR/Cas9基因组编辑能够在空间和时间上得到调节。
