Plv5 cas9 blast

The production of lentivirus for MC38‐Coro1a or MC38‐Coro1a‐K233R overexpression was carried out as follows. HEK293 cells were seeded into one 10 cm dish. The following day, the cells were transfected with 5 μg of plvx‐mCherry‐Coro1a‐Flag or 5 μg of plvx‐mCherry‐Coro1a‐K233R‐Flag, 3.75 μg of psPAX2 (gag, pol) and 1.25 μg of pMD2.G using 20 μl of JetPEI. Viral supernatants were collected 48 h after transfection and filtered through 0.45 μm PVDF filters. For initial lentiviral transduction, MC38 cells were infected with the appropriate virus in six‐well plates in the presence of 10 μg ml⁻¹ polybrene and centrifuged at 1500 × g for 2 h at 32°C. After 24 h, single mCherry⁺ cells were sorted into 96‐well plates with a Beckman MoFlo Astrios EQ (Beckman). The levels of endogenous and overexpressed proteins were then verified by WB.
For Coro1a knockout, HEK293 cells were seeded into 12‐well plates. The following day, the cells were cotransfected with plv5‐Cas9‐Blast (Merck) and U6‐gRNA: hPGK‐puro‐2A‐tBFP (Merck) including a specific gRNA sequence listed in Table S1. After 24 h, single BFP⁺ cells were sorted into 96‐well plates with a Beckman MoFlo Astrios EQ. The levels of endogenous and overexpressed proteins were then verified by WB.