Crispr cas9 genome editing

Human TET2, p53, Atg16L1, Beclin1, LC3A, LC3B, p62 were cloned from HEK293T cells using reverse transcription PCR. p53 mutants (R175H, K305A and L348A+L350A) were constructed by using the QuikChange site-directed mutagenesis kit (Catalog #200523) from Agilent Inc.. For transient expression in mammalian cells, the cDNA encoding human TET2 was cloned into a modified pcDNA3-Flag-Dest-B vector. The CRISPR/Cas9 genome editing construct PX459 (62988; Addgene) was used for sgRNA-directed knockout of TET2, p53, Atg16L1, Beclin1, LC3, p62.

TET2 sgRNA-1: caccgTGGAGAAAGACGTAACTTCG

TET2 sgRNA-2: caccgCAGGACTCACACGACTATTC

p53 sgRNA-1: caccgCCATTGTTCAATATCGTCCG

p53 sgRNA-2: caccgCCCCGGACGATATTGAACAA

Atg16L1 sgRNA: caccgAGATGTGGCGCTTCCAGCGG

Beclin1 sgRNA: caccgGGACACGAGTTTCAAGATCC

p62 sgRNA : caccgCACCGTGAAGGCCTACCTTC

LC3 sgRNA: caccgACCTCCTTACAGCGGTCGGC

The sgRNA-containing plasmids were transfected into HEK293T cells and subjected to puromycin selection (2 μg ml⁻¹) for 2–4 days. Survival clones were then seeded into 96-well plates and later expanded to 24-well plates for maintenance in the presence of 1 μg ml⁻¹ puromycin. The gene disruption was confirmed by western blotting. All the plasmid information was listed in Table S2.