条件单载体 CRISPR/SaCas9 病毒在成年小鼠神经系统中的高效诱变作用。
Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient Mutagenesis in the Adult Mouse Nervous System.
机构信息
Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, WA 98195, USA; Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.
Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.
出版信息
Cell Rep. 2020 Mar 24;30(12):4303-4316.e6. doi: 10.1016/j.celrep.2020.02.092.
Mice engineered for conditional, cell type-specific gene inactivation have dominated the field of mouse genetics because of the high efficiency of Cre-loxP-mediated recombination. Recent advances in CRISPR/Cas9 technologies have provided alternatives for rapid gene mutagenesis for loss-of-function (LOF) analysis. Whether these strategies can be streamlined for rapid genetic analysis with the efficiencies comparable with those of conventional genetic approaches has yet to be established. We show that a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA (sgRNA) are as efficient as conventional conditional gene knockout and can be adapted for use in either Cre- or Flp-driver mouse lines. The efficacy of this approach is demonstrated for the analysis of GABAergic, glutamatergic, and monoaminergic neurotransmission. Using this strategy, we reveal insight into the role of GABAergic regulation of midbrain GABA-producing neurons in psychomotor activation.
通过 Cre-loxP 介导的重组，条件性、细胞类型特异性基因敲除的小鼠已成为小鼠遗传学领域的主流，因为其具有高效性。CRISPR/Cas9 技术的最新进展为基因失活（LOF）分析的快速基因诱变提供了替代方案。这些策略是否可以与传统遗传方法相媲美，实现快速遗传分析的效率，还有待确定。我们表明，单个腺相关病毒（AAV）载体包含一个重组酶依赖性金黄色葡萄球菌 Cas9（SaCas9）和一个单指导 RNA（sgRNA），与传统的条件性基因敲除一样高效，并且可以适应 Cre 或 Flp 驱动的小鼠系使用。该方法用于分析 GABA 能、谷氨酸能和单胺能神经传递的功效。使用这种策略，我们揭示了中脑产生 GABA 的神经元中 GABA 调节对精神运动激活的作用。
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