Synthetic genetic circuits have revolutionised our capacity to control cell viability by conferring microorganisms with programmable functionalities to limit survival to specific environmental conditions. Here, we present the GenoMine safeguard, a CRISPR-Cas9-based kill switch for the biotechnological workhorse that employs repetitive genomic elements as cleavage targets to unleash a highly genotoxic response. To regulate the system's activation, we tested various circuit-based mechanisms including the digitalised version of an inducible expression system that operates at the transcriptional level and different options of post-transcriptional riboregulators. All of them were applied not only to directly control Cas9 and its lethal effects, but also to modulate the expression of two of its inhibitors: the AcrIIA4 anti-CRISPR protein and the transcriptional repressor TetR. Either upon direct induction of the endonuclease or under non-induced conditions of its inhibitors, the presence of Cas9 suppressed cell survival which could be exploited beyond biocontainment in situations where further CRISPR genome editing is undesirable.
Synthetic genetic circuits have revolutionised our capacity to control cell viability by conferring microorganisms with programmable functionalities to limit survival to specific environmental conditions. Here, we present the GenoMine safeguard, a CRISPR-Cas9-based kill switch for the biotechnological workhorse that employs repetitive genomic elements as cleavage targets to unleash a highly genotoxic response. To regulate the system's activation, we tested various circuit-based mechanisms including the digitalised version of an inducible expression system that operates at the transcriptional level and different options of post-transcriptional riboregulators.
Internal tandem duplication mutations of FLT3 (FLT3/ITD) confer poor prognosis in AML. FLT3 tyrosine kinase inhibitors (TKIs) alone have limited and transient clinical efficacy thus calling for new targets for more effective combination therapy. In a loss-of-function RNAi screen, we identified NOTCH4 as one such potential target whose inhibition proved cytotoxic to AML cells, and also sensitized them to FLT3 inhibition.
Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages.
Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes simplex virus type 1 (HSV-1) has been proposed as a potential treatment for cancer and was moved to phase III clinical trials. Previous studies showed that design of OV therapy combined with p53 gene therapy increases the anti-cancer activities of OVs.
School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering (Ministry of Education), Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, 300350, P. R. China.
CRISPR/Cas9-based gene therapy and photodynamic therapy both show promise for cancer treatment but still have their drawbacks limited by tumor microenvironment and long treatment duration. Herein, CRISPR/Cas9 genome editing and photodynamic strategy for a synergistic anti-tumor therapeutic modality is merged. Chlorophyll (Chl) extracted from natural green vegetables is encapsulated in Pluronic F127 (F127) micelles and Histidine-tagged Cas9 can be effectively chelated onto micelles via metal coordination by simple incubation, affording Cas9-Chl@F127 micelles.
