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As a new generation of genome editing technology, the CRISPR/Cas9 system provides a revolutionary tool for life science research and opens up new opportunities for biotechnology applications in the fields of medicine, agriculture and industry.
But in many eukaryotic organisms, the establishment and application of the CRISPR/Cas9 system is greatly restricted by the lack of efficient expression of guided RNA.
is an important industrial microorganism, its products citric acid, enzyme preparations and other uses in food, medicine, chemical and other fields are widely used, but at present, it is still impossible to achieve the high-efficiency genome editing of heludonium, limiting its potential for engineering applications.
recently, the Systems Biology Center and researcher Zheng Ping, led by Sun Yibin, a researcher at the Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, led a team of systems and synthetic biotechnology research team, for the first time in the important industrial microorganism black citform, with ribosome 5S rRNA-based sgRNA-based expression, developed a new 5S rRNA-based high-efficiency CRISPR/Cas9 system, so that Cas9 for the black citriceclic genome fixed-point cutting efficiency of up to 100%.
Figure 1 applies different guide RNA expression strategies to the CRISPR/Cas9 system mediated the inactivation of the black-cranked albA gene.
(A) the genome editing process diagram of albA gene inactivation mediated by the CRISPR/Cas9 system; (B) 5S rRNA gene initiates core sequence identification of guide RNA expression; and (C) the effect of different promoter strategies on the efficiency of albA gene inactivation.
has established a black-cranked high-efficiency genome editing toolkit, with 40 bp of short homologous arm donor DNA that can be easily edited by unit points, multi-point gene knock-in, and large fragment DNA knockout long to 48 kb.
the new CRISPR/Cas9 system effectively solves the active expression of black buckitus sgRNA, breaks through the bottleneck of high-efficiency editing of the black crucible genome, provides strong technical support for understanding the biological molecular basis of the potential of the black crankmold industry, further enhances its industrial application performance, and develops new cell factories and new products. At the same time,
, due to the high conservatism of ribosome 5S rRNA, its 5S rRNA gene can be quickly found in any living organism, based on 5S rRNA guide RNA expression strategy, to solve the eyre ticor RNA expression problems, the realization of CRISPR/Cas9 genome editing system in various eyre organisms to establish and apply a new way of universal thinking.
the research was supported by the NSFC Youth and Face Project and the Chinese Academy of Sciences International Talent Program, and the results have been published in the journal ACS Synthetic Biology. Zheng Xiaomei, an assistant researcher at Tianjin Institute of Engineering and
, is the first author of the paper.
.