Efficient and reversible Cas13d-mediated knockdown with an all-in-one lentivirus-vector

FIGURE 1. pLenti-TRE-CasRx-U6 expresses CasRx in a tightly-inducible manner. (A) Schematic representation of plenti-TRE-CasRx. (B) A total of 293 T cells were transfected with pLenti-TRE-CasRx; 24 h later, the cells were treated with 1 μg/ml Doxycycline for 24 h. Shown are WB results. First lane shows Donor cell. (C) HLF cells were transduced with a virus expressing pLenti-TRE-CasRx. Cells were selected with 1 μg/ml puromycin for 5 days and then treated with 1 μg/ml Doxycycline for 24 h. Shown are WB results. First lane shows Donor cell. (D) HLF cells were transduced with a virus expressing pLenti-TRE-CasRx; 48 h later, the cells were treated with 1 μg/ml Doxycycline for 24 h. Shown are immunofluorescence results. Nuclei were counter-stained with DAPI. Scale bar: 50 μm. (E) HLF cells were transduced with different volumes of virus suspension and then selected with puromycin. Cell proliferation during 4 days was counted and normalized to the cells infected with 2 ml virus which was confirmed to express HA-CasRx at about 100% with immunofluorescence.

FIGURE 2. Knockdown can be induced in a Doxycycline dosage-dependent manner. (A) 293 T cells were transfected with pLenti-TRE-CasRx-scram. Cells were treated with 1 μg/ml Doxycycline 24 h hours later for the indicated time. Shown are WB results. (B) Ctrl or KDM1B-knockdown (KD) cells were seeded into a 6-well plate. Knockdown was induced with 1 μg/ml doxycycline. Shown is the relative cell proliferation during 7 days normalized to the control (Ctrl) group. “NS” denotes “not significant” as examined with a two-sided unpaired Welch’s t-test. (C) HLF cells were transduced with a virus expressing pLenti-TRE-CasRx-KDM1B-sgRNA. Cells were then selected with 1 μg/ml puromycin for 5 days and treated with 1 μg/ml Doxycycline for different times. Shown are the WB results. (D) HLF cells were transduced with a virus expressing pLenti-TRE-CasRx-DOT1L-sgRNA. Cells were then selected with 1 μg/ml puromycin for 5 days and treated with 1 μg/ml Doxycycline for different times. Shown are the WB results. (E) HLF cells were transduced with a virus expressing pLenti-TRE-CasRx-scramble. Cells were then selected with 1 μg/ml puromycin for 5 days. Afterward, cells were treated with 1 μg/ml Doxycycline for…

FIGURE 3. pLenti-TRE-CasRx-U6-gRNA can achieve reversible knockdown. (A) HLF cells transduced with a virus expressing pLenti-TRE-CasRx-scram were selected with 1 μg/ml puromycin for 5 days and then treated with 25 μg/ml cycloheximide for the indicated time. Shown are WB results. (B) HLF cells transduced with a virus expressing pLenti-TRE-CasRx-scram were selected with 1 μg/ml puromycin for 5 days and then treated with 1 μg/ml Doxycycline for 4 days. Cells were then cultured in a fresh medium without doxycycline for the indicated time. Shown are WB results.

FIGURE 4. Different types of genes can be efficiently knocked-down. (A) HLF cells were transduced with the pLenti-TRE-CasRx-METTL3-sgRNA virus. Cells were then selected with 1 μg/ml puromycin for 5 days. Afterward, cells were treated with 1 μg/ml Doxycycline for 4 days. The left panel shows the WB results. The right panel shows the densitometric analysis of the left panel. (B) HLF cells were transduced with the pLenti-TRE-CasRx-FKBP8-sgRNA virus. Cells were then selected with 1 μg/ml puromycin for 5 days. Afterward, cells were treated with 1 μg/ml Doxycycline for 4 days. The left panel shows the WB results. The right panel shows the densitometric analysis of the left panel. (C) HLF cells were transduced with the pLenti-TRE-CasRx-OGT-sgRNA virus. Cells were then selected with 1 μg/ml puromycin for 5 days. Afterward, cells were treated with 1 μg/ml Doxycycline for 4 days. The left panel shows the WB results. The right panel shows the densitometric analysis of the left panel. (D) HLF cells were transduced with the pLenti-TRE-CasRx-MALAT1-sgRNA virus. Cells were then selected with 1 μg/ml puromycin for 5 days. Afterward, cells were treated with 1 μg/ml Doxycycline for 4 days. Shown are the…

FIGURE 5. Genes can be efficiently knocked-down in different cell types. (A–D) Cells were transduced with a virus expressing pLenti-TRE-CasRx-KDM1B-sgRNA. Cells were then selected with 1 μg/ml puromycin for 5 days. Afterward, cells were then treated with 1 μg/ml Doxycycline for 4 days. Shown are WB results. (E,F) Cells were transduced with a virus expressing pLenti-TRE-CasRx-Mettl3-sgRNA. Cells were then selected with 1 μg/ml puromycin for 5 days. Afterward, cells were treated with 1 μg/ml Doxycycline for 4 days. Shown are WB results.

FIGURE 6. Indispensable genes could be efficiently knocked-down in cells hard to transduce. (A) Same number of MV4-11 cells were transduced with a virus expressing constitutive DOT1L-sgRNA or control sgRNA. Cells were then selected with 1 μg/ml puromycin for 5 days. Shown is the relative fold of proliferation after selection as compared to the group without puromycin selection. (B) Same number of MV4-11 cells were transduced with a virus expressing pLenti-TRE-CasRx-DOT1L-sgRNA or control vector. Cells were then selected with 1 μg/ml puromycin for 5 days. Afterward, cells were treated with 1 μg/ml Doxycycline for 7 days. The left panel shows the relative fold of proliferation after selection as normalized to the cells without Doxycycline induction. The right panel shows WB results.

FIGURE 7. Genes can be efficiently knocked-down in vivo. HLF cells expressing EZH2-sgRNA or control vector was inoculated subcutaneously into nude mice. Gene knock-down was induced with Doxycycline in drinking water. (A) Growth curve of the xenograft tumors. (B) Western Blot results for four representative tumors.