CRISPR/Cas9 system is an adaptive immune defense formed in the long-term evolution of bacteria and archaea, as it can also direct the degradation of virus and foreign nucleic acids. By delivering the Cas9 nuclease and appropriate guide RNAs into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. Moreover, the nuclease-deactivated dCas9 is also proved to be effective in the regulation of gene expression and the mark of specific DNA sites. Therefore, by using layered gene circuits and precisely regulating dCas9, several strategies have been developed to engineer modular CRISPR/Cas systems.
However, the application of CRISPR/Cas therapeutic circuits is still challenging due to the restrictive cargo size of existing viral delivery vehicles.
Lately, breakthroughs in reducing the size of synthetic circuits have been made by Zhen Xie’s team of MOE Key Laboratory of Bioinformatics and Bioinformatics Division, TNLIST, Tsinghua University. Xie’s team published a research article entitled ‘Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells’ in < Nature Communications > on 3rd Oct, 2016. In this study, Xie’s team first verified that dCas9 could be split and reconstituted primarily in human cells. Then they engineered sensory switches by exchanging split dCas9 domains, allowing differential regulations on one gene, or activating two different genes in response to cell-type specific microRNAs. In general, their work provided evidence in precise regulation of Cas9/dCas9 activity in different types of cells and inspired new strategies to the precise control of Cas9/dCas9 gene edit toolkit.
Xie’s work on precise regulation of dCas9 functions by engineering sensory switches is another important contribution in synthetic biology following the publication of modular construction of mammalian gene circuits using TALE transcriptional repressors in <Nature chemical biology> 2015.
Prof. Xie Zhen is the correspondence author of this paper. PhD. candidate Dacheng Ma from the Department of Automation, Tsinghua University, is the first author and M.S. candidate Shuguang Peng from the same department contributed equally to this work. The research has been supported by the National Key Basic Research Program of China (‘973’ project) and the Basic Research Program of Tsinghua National Lab for Information Science and Technology.