Zhibo Wang, first author of “A Reverse Chromatin Immunoprecipitation technique based on the CRISPR dCas9 system”
Current Position: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, doctoral candidate
Education: Successive postgraduate and doctoral programs of study in State Key Laboratory of Tree Genetics and Breeding of Northeast Forestry University in 2015-2022
Non-scientific Interests: Sports, hiking and music
I study at State Key Laboratory of Tree Genetics and Breeding of Northeast Forestry University now. I studied on physiological and molecular biology of forest resistance under the guidance of Pro. Yucheng Wang since entrance. I study in function analysis of transcription factors related with stress resistance and mechanism research of protein post-translational modifications regulating stress resistance in Betula platyphylla. In this research, we explored a system called R-ChIP-dCas9 based on the characteristic that the CRISPR-dCas9 system can specifically recognize the DNA site and without the nucleolytic activity. In this system, dCas9 was fused with Strep-Tag II. Transient transformation was performed for the expression of dCas9-Strep-DNA complex in birch plants. The dCas9-Strep-DNA complex was crosslinked and purified using StrepTactin beads for obtaining target DNA fragments. The proteins binding to the target genomic DNA region were identified using mass spectrometry. This technology can capture the DNA region of interest in the genomes of plant species quickly and specifically, can be adapted to various plant species and does not depend on the availability of a stable transformation system. Therefore, this method might provide new thoughts for identifying proteins bound to genomic DNA.
论文：A Reverse Chromatin Immunoprecipitation technique based on the CRISPR dCas9 system
我目前就读于东北林业大学林木遗传育种国家重点实验室。从2015年入学以来，始终在王玉成教授课题组进行林木抗逆生理与分子生物学研究。目前主要从事白桦抗逆相关转录因子功能解析及其蛋白质翻译后修饰对抗逆调控机制等方面的研究。本研究中，我们利用dCas9具有结合目的DNA且不会对DNA进行切割的特点，开发了R-ChIP-dCas9系统。将dCas9蛋白与Strep-Tag II融合，通过瞬时转化技术将其转入植物中，在植物体内形成dCas9-Strep-DNA复合体，然后用甲醛进行DNA和蛋白的交联，交联后Strep-Tag II蛋白纯化磁珠纯化目的DNA片段，进而获得了相应的DNA-binding protein，并通过质谱分析鉴定出目的DNA的结合蛋白。该方法具有高效、快捷、可靠等优点且不依赖于稳定转化，为鉴定基因的上游调控因子提供了新的方法和思路。