Dual-luciferase reporter analysis
For luciferase report assays, HEK293T cells were seeded in 96-well plate
(1×104 cells/well) 24 hours before plasmid
transfection. The Cas13b-gRNA or Cas13d-gRNA plasmids with
pmirGLO-REPORT™ vectors were co-transfected into cells using
lipofectamine 8000 (Beyotime), fresh medium was added 24 hours after
transfection. Cells were assayed 48 hours after transfection using
Dual-Luciferase Reporter Assay System (Beyotime). Briefly, the cells
were lysed with cell lysis buffer (80 μl/well) on ice for 30 min. 30 μl
of cell lysis was mixed with 30 μl luciferase substrate in the dark, and
the luciferase emission was read by a single-tube chemiluminescence
detector. The renilla luciferase substrate was added to the mixture and
the renilla luciferase luminescence was read again. Renilla luciferase
activity was used as an internal reference.