The CRISPR-Cas9 system successfully knocked out the exogenic dsRFP gene from the cotton genome.

Recently, our cotton research team has made important progress in the application of CRISPR/Cas9 technology in heteroglytic four-body cotton, and the results are published online in Plant Biotechnology Journal, a leading academic journal in botany, entitled "High efficient multi-sites genome editing in alltraoteploid cotton" (Gossypium hirsut) AND CRISPR/Cas9 system.
relying on the team's efficient genetic transformation platform, this study expands the application of CRISPR technology in polyplytic crops and provides important technical means for the analysis of cotton functional genes.
co-authors of this paper are Ph.D. student Wang Pengcheng and master's student Zhang Jun, Professor Zhang Xianlong and Professor Jin Shuangxiao as co-authors of the paper.
successful application in bio-organisms in 2013, CRISPR/Cas9 system has become the focus of life science research and the most popular genome editing system in recent years.
system has the characteristics of strong stability, high efficiency and simple operation.
using CRISPR/Cas9 technology to successfully achieve genome-level gene knockout, gene transcription regulation and modification.
the current technology is widely used in animal cells, plants in rice, acetic mustard, corn and other model crops are more used, and in polypolycular plants, such as cotton, rape in less applications.
The reason is that the cotton currently grown in production is a heterogenous four-fold body, there are 52 chromosomes, the genome is large and complex (2.5 Gb, equivalent to 20 times the size of the amoeba genome, 6 times the rice genome), the same repeated sequence (about 70%), the same gene also has multiple copies, it is difficult to obtain effective single gene mutants.
The current conventional method of genetic function research in cotton is RNAi technology, which often causes the silent expression of the same gene (gene family) to produce unexpected dnotypes, while RNAi technology often silent on the target gene is incomplete, the silent effect in future generations is unstable, and even disappear, so it is imperative to develop a new functional genomic research method in cotton.
china Agricultural University cotton team from 2013 onwards, in cotton began the research work of gene editing, including the construction of a TALEN vector for the cotton genetic conversion system and three sets of CRISPR-Cas9 system.
The carrier used in this study is the third SET of CRISPR-Cas9 system used in this laboratory, which has been modified for the existing CRISPR/Cas9 expression carrier in rice, using the U6 starter of cotton endogenous source and suitable antibiotic screening markers.
used a material that had obtained the exogenated reported gene DsRed2 (Red Fluorescent Protein Gene) to perform a secondary transformation of the receprotes, resulting in the knockout of two target points at the same time, and the red fluorescence of the regenerative plant disappeared completely.
In addition, the in-line cotton chlorophyl synthesis-related gene GhCLA1 for the target design of three pairs of sgRNA also successfully achieved gene knockout, the editing efficiency of each target site reached 66.7% to 100%, the regenerative plant produced a significant alemerization seed physinotype.
genetic analysis of T0 plants and their offspring showed that the target gene could be mutated without off-target effects, and that the mutation could be passed on steadily to future generations.
The innovation of this study is to fully combine the second generation of high-fluncy sequencing technology to achieve rapid identification of a large number of regenerative plant mutation sites, greatly improve the efficiency of mutation identification of genetically modified plants, and create a cotton gene mutation bank and functional genome research in order to achieve CRISPR/Cas9 system to lay a solid foundation.
At present, this efficient cotton gene editing vector has been distributed through cooperative research to more than 10 domestic and foreign scientific research institutions, including the Australian Federal Scientific and Industrial Research Organization (CSIRO), Zhejiang University, Nanjing Agricultural University, Huahua University of Science and Technology, Central China Normal University, Southwest Agricultural University, Shandong Agricultural University, Ashiko University, China Cotton Institute and other domestic and foreign scientific research institutions, to jointly promote the genome editing of cotton.
research has received strong support from the 13th Five-Year National Key Research and Development Fund and the Special Fund for GeneticAlly Modified Products.
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