Fig. 1

CRISPR/Cas13d mediated gene knockdown in T cells. a Reversion of tonic signaling phenotype in CAR T cells. The tonic signaling CAR T cell model overexpresses three inhibitory receptors (LAG3, PD-1, TIM-3) which lead to a T cell exhaustion phenotype. By transducing these cells with an array of guide crRNAs, the respective genes are knocked down by target specific Cas13d mediated RNA degradation. As a result, the exhaustion markers are no longer expressed, leading to improved T cell function such as cell expansion, memory, fitness and even tumor clearance. b Drug-inducible gene knockdown. A destabilization domain, fused to Cas13d leads to proteasomal degradation of the effector, disabling gene knockdown in the ground state. By addition of trimethoprim, the domain is stabilized and Cas13d cleavage is enabled. The system is reversible and can be utilized to dynamically regulate gene knockdown of T cell receptors by administration or withdrawal of the drug. c Potential MEGA-based T cell diagnostics. Dysregulated T cells could be extracted, transduced with the multiplexed guide array and screened for abnormally expressed genes. Afterwards, the cells could be transduced with guides targeting the identified genes, restoring physiological T cell function. Together with either inducible or permanent gene knockdown or in combination with other drugs, an individualized treatment plan could be implemented