Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice.
Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice.
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DOI：
10.1038/s41434-021-00224-2
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发表时间：
2021-11
期刊：
影响因子：
5.1
通讯作者：
Lo SC
中科院分区：
文献类型：
--
作者：
Hana S;Peterson M;McLaughlin H;Marshall E;Fabian AJ;McKissick O;Koszka K;Marsh G;Craft M;Xu S;Sorets A;Torregrosa T;Sun C;Henderson CE;Lo SC
CRISPR-Cas系统已经成为研究正常和患病中枢神经系统（CNS）生成遗传模型的有力工具。在非分裂神经元中已经成功地证明了特定位点的靶向基因破坏。尽管简单、高特异性和低成本，crispr介导的基因敲除在体内的效率会受到许多参数的显著影响。在这里，我们使用CRISPR-Cas9破坏神经元特异性基因NeuN，并优化关键参数，在出生后小鼠的中枢神经系统中实现有效的基因敲除。三个细胞系和两个原代神经元培养物被用来验证含有不同间隔和支架序列的单导rna （sgRNA）对NeuN的破坏。该分类确定了体外和体内系统中具有最高NeuN破坏的最佳sgRNA设计。为了提高CRISPR的效率，AAV-PHP。B是一种具有优越神经元转导的载体，通过新生儿脑室内（ICV）注射在Cas9小鼠中传递该sgRNA。这种方法导致转导细胞中的双等位基因指数达到99.4%，导致皮层、海马和脊髓中总NeuN蛋白减少70%以上。这项工作有助于优化crispr介导的基因敲除，并将有利于基础和临床前研究。
CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.
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影响因子：
48
作者：
Anderson KR;Haeussler M;Watanabe C;Janakiraman V;Lund J;Modrusan Z;Stinson J;Bei Q;Buechler A;Yu C;Thamminana SR;Tam L;Sowick MA;Alcantar T;O'Neil N;Li J;Ta L;Lima L;Roose-Girma M;Rairdan X;Durinck S;Warming S
通讯作者：
Warming S
影响因子：
12.3
作者：
Dang Y;Jia G;Choi J;Ma H;Anaya E;Ye C;Shankar P;Wu H
通讯作者：
Wu H
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
13.6
作者：
Gaj T;Ojala DS;Ekman FK;Byrne LC;Limsirichai P;Schaffer DV
通讯作者：
Schaffer DV
影响因子：
25
作者：
Chan KY;Jang MJ;Yoo BB;Greenbaum A;Ravi N;Wu WL;Sánchez-Guardado L;Lois C;Mazmanian SK;Deverman BE;Gradinaru V
通讯作者：
Gradinaru V