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Genome Editing As an Approach to the Study of in Vivo Transcription Reprogramming.
Doklady Biochemistry and Biophysics ( IF 0.8 ) Pub Date : 2020-04-27 , DOI: 10.1134/s1607672920010147
Y Y Silaeva 1 , V A Kalmykov 1 , E A Varlamova 1, 2 , E N Korshunov 1 , D S Korshunova 1 , M V Kubekina 1 , A A Shtil 1, 2 , I B Roninson 1, 3 , A V Deykin 1, 4
Affiliation  

Abstract

CDK8-mediated transcriptional reprogramming is essential for an extensive gene expression. Constitutive knockouts of the cdk8 gene are lethal at the morula stage. For modeling transcriptional reprogramming in an adult organism, we investigated the possibility to attenuate the CDK8 kinase activity with a F97G mutation in exon 3 of the cdk8 gene. According to preliminary experimental data, this mutation should lead to a decrease in CDK8 kinase activity. To edit the genome of laboratory mice, the CRISPR/Cas9 technology was used, in which the introduction of a double-stranded gap occurred at a distance of 128 nucleotide pairs from the planned site of the introduced mutation. To introduce the mutation, a matrix for homologous repair was used as part of plasmid DNA, with homologous arms 903 and 484 bp in the 5'–3' region from the point of double-stranded rupture, respectively. As a result, mice with site-specific target mutations in exon 3 of the cdk8 gene were obtained. We for the first time demonstrated a high efficacy of the mutation 128 bp apart from the site of double-strand break. Viable animals with the F97G mutation in the catalytic domain of CDK8 kinase were obtained for the first time. The resulting cdk8 mutant mice will be used in subsequent studies to simulate the processes involving transcription reprogramming.


中文翻译:


基因组编辑作为体内转录重编程研究的一种方法。


 抽象的


CDK8 介导的转录重编程对于广泛的基因表达至关重要。 cdk 8 基因的组成型敲除在桑葚胚阶段是致命的。为了模拟成年生物体中的转录重编程,我们研究了通过cdk 8 基因外显子 3 中的 F97G 突变减弱 CDK8 激酶活性的可能性。根据初步实验数据,这种突变应该会导致CDK8激酶活性降低。为了编辑实验室小鼠的基因组,使用了 CRISPR/Cas9 技术,其中双链缺口的引入发生在距引入突变的计划位点 128 个核苷酸对的距离处。为了引入突变,使用同源修复基质作为质粒 DNA 的一部分,同源臂分别位于距双链断裂点的 5'–3' 区域 903 和 484 bp。结果,获得了cdk8基因外显子3具有位点特异性靶突变的小鼠。我们首次证明了除双链断裂位点外128 bp突变的高效性。首次获得CDK8激酶催化域F97G突变的活动物。由此产生的cdk 8突变小鼠将用于后续研究,以模拟涉及转录重编程的过程。
更新日期:2020-04-27
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