The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems. Next, we show that introduction of split-Csy4 into human cells caused a substantially reduced extent in perturbation of the endogenous transcriptome when directly compared to full-length Csy4. Inspired by these results, we went on to use such split-Csy4 module to engineer inducible CRISPR- and translation-level gene switches regulated by the FDA-approved drug grazoprevir. This work provides valuable resource for Csy4-related biomedical research and discusses important issues for the development of clinically eligible regulation tools.
The fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973) has been considered a good chassis candidate for “microbial cell factory” as it can perform oxygenic photosynthesis and its doubling time can be as short as 1.9 h. However, the limited genetic tools currently restrict its further research and application efforts using synthetic biology approaches. In this study, a series of genetic tools were systematically developed and optimized for Synechococcus 2973. First, the introduction of Tfp pilus assembly protein encoding gene pilN into Synechococcus 2973 successfully recovered its natural transformability, which greatly simplified the DNA transformation process. Second, a series of promoters with different strengths were evaluated and the super-strong promoters including P cpc560 from Synechocystis sp. PCC 6803, native P psbA2 and P psbA3 of Synechococcus 2973 were found with the highest activity of β -galactosidase among those evaluated by miller values. Some promoters related to photosystems (i.e., P psbA2 , P psbA3 , P 6803psbA2 and P cpc560 ) were also demonstrated to be induced by high intensity of light. Third, three lactose induction systems were evaluated, among which P lac combined with lacI q showed the best application prospect with great induction capacity, low leakage and middle induced expression. Fourth, the translational on riboswitch theoE* , the transcriptional off riboswitches theo/yitJ and xpt(C74U)/metE and an artificial inducing system combining theoE* with T7 RNA polymerase were successfully developed and characterized in Synechococcus 2973. Finally, by using T7 induction system to control the expression of both small RNA and chaperone Hfq, a small RNA regulatory tool was developed and optimized to be a strictly inducible off system for gene regulation in Synechococcus 2973. The work here presented valuable genetic toolboxes necessary for metabolic engineering and synthetic biology research in Synechococcus 2973, which will facilitate the future application of the fast growing cyanobacterial chassis.
危险品化学品经营许可证（带存储）