Abstract

BACKGROUND: Ever since the technology was discovered, inducible CRISPR systems have become a highly sought-after tool. Several groups have proposed different strategies to achieve inducible CRISPR-based genome-editing control, which goes from splitting Cas9 into two parts to be later dimerized by a compound, until the regulation of the promoter of Cas9 and sgRNA with RNA-polymerase repressors, such as the tet system. METHODS: In this study, we construct and evaluate a doxycycline-dependent combinatorial inducible CRISPR system, encompassing the tet repressor, Cas9, and a fluorescent reporter in a single plasmid. The system enables conditional sgRNA expression through a double tet operator, also present in the plasmid. The tool was successfully tested in vitro to knock out a pathogenic recombinant human autoantibody (h-IgG-MAb) that binds to the acetylcholine receptor (Clone B12L). RESULTS: A sgRNA (named B12L-Ko-gRNA), targeting the variable region of the heavy chain (B12L-VH), was introduced with the all-in-one CRISPR tool. Within one month, no significant expression of the B12L-Ko-gRNA was observed, but when doxycycline was added, secreted levels of B12L-h-IgG-MAb decreased similar to 94% when compared to non-target (NT-gRNA), suggesting efficient doxycycline-induced CRISPR-knockout of B12L-VH. However, in a long-term analysis (>3 months), small but significant sgRNA leaking was observed, affecting the target gene expression, evidenced by a decrease to similar to 60% in the B12L-VH reported expression compared to >90% in the NT-sgRNA. CONCLUSIONS: The doxycycline-sensitive tool developed in this study was effective in the short-term (within 1 month) with insignificant sgRNA expression in absence of doxycycline. For long-term applications, the system could be combined with additional expression-controls, such as the split-Cas9 strategy.