Mouse anti β actin

Cell lysates were subjected to Western blot analysis with the following antibodies and dilutions: rabbit anti-PCGF1 (1:4000; Abcam, USA), mouse anti-β-Actin (1:4000; CWBIO, China), rabbit anti-AKT (1:4000, Abcam, USA), rabbit anti-pAKT (1:4000, Abcam, USA), rabbit anti-GSK-3β (1:4000, Abcam, USA), rabbit anti-c-Myc (1:4000, Abcam, USA), and rabbit anti-CyclinD1 (1:4000, Abcam, USA). The signals were amplified by HRP-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA), and detected via ECL Plus (Amersham Pharmacia Biotech, USA). The acquired images were then analyzed on a computer using Image J software.

MLH1 protein expression in screened monoclonal cells was assessed using Western blot (WB) and next‐generation sequencing (NGS). For WB, the monoclonal cells were lysed with 500 µl of RIPA lysis buffer (Solarbio, Beijing, People's Republic of China). Lysates were separated by SDS‐PAGE with an 8% separation gel and 5% concentration gel. After transferring to polyvinylidene difluoride (PVDF) membranes, blots were probed with the primary antibodies (rabbit anti‐MLH1 (BD Bioscience, Franklin Lakes, New Jersey; 1:100000) and mouse anti‐β‐actin (CWBio, Beijing, People's Republic of China; 1:5000) polyclonal antibodies), followed by secondary antibodies (mouse anti‐rabbit IgG (Santa Cruz Bio Inc., Dallas, Texas; 1:5000) and goat anti‐mouse IgG (Santa Cruz Bio Inc.; 1:5000)). GM12878Cas9 and HCT116 cell lines were used as positive and negative controls, respectively. For NGS, genomic DNA was extracted from monoclonal cells using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and 80–120 ng of DNA was used as input for the library preparation. A pool of oligos specific to 523 genes (TruSight Oncology 500 Kit, Illumina, Santiago, Chile) was used to prepare DNA libraries for sequencing on the Illumina NextSeq 550 platform. All procedures were performed following the manufacturer's instructions. Data analysis was performed using the TSO500 module in the commercial software (Illumina).

The human brain hippocampus in each group was excised. The samples were homogenized in T‐PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, USA) containing Protein Phosphatase Inhibitor (Solarbio, Beijing, China) and phosphatase inhibitor Cocktail (CWBio, Beijing, China). The lysates were then homogenized and centrifuged (12,000g for 15 min at 4°C). The protein samples were then separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, USA). The membranes were blocked in 5% (w/v) BSA for 1 h at room temperature and incubated with primary antibodies (rabbit anti‐DLL3, 1:1000, Cat: ab63707; Abcam; rabbit anti‐Notch2, 1:1000, Cat: D67C8; Cell Signal Technology; rabbit anti‐BMP6, 1:1000, Cat: D2V5Z; Cell Signal Technology; rabbit anti‐ID2, 1:1000, Cat: D39E8; Cell Signal Technology; mouse anti‐β‐actin, 1:1000, Cat: CW0096; CWBio) overnight at 4°C, followed by incubation with corresponding secondary antibody for 1 h at room temperature. The bands were scanned with Tanon 5800 Luminescent Imaging Workstation (Tanon Science & Technology Co., Ltd. Shanghai, China) by High‐sig ECL Western Blotting Substrate (Solarbio, Beijing, China). The band intensity was measured by Image J software (National Institutes of Health, Bethesda, MD).