sgRNA targeted the exon 3 of PUMA gene, which contains sequence code for BH3 domain of PUMA. Two crRNAs introduced into lentiviral vectors (pLentiCRISPR-E, Addgene #78852) which contains eSpCas9 and puromycin cassette.

Guide1 DNA (forward, 5’-CACC GGCGGGCGGTCCCACCCAGG-3’; reverse, 5’-AAAC CCTGGGTGGGACCGCCCGCC-3’) and Guide 2 DNA (forward, 5’-CACC GCCGCTCGTACTGTGCGTTG-3’; reverse, 5’-AAAC CAACGCACAGTACGAGCGGC-3’) were annealed and ligated to the restriction enzyme-cut plasmid by T4 ligase. Stbl3 strain (Invitrogen C7373-03) was transformed by the guides-containing plasmids. LB-amp plates were streaked and incubated on a shaker at 37 °C overnight. The bacterial colonies were selected and mixed up with LB (Terrific Broth) and 100 μg/mL ampicillin, and were incubated on a shaker at 37 °C overnight. Plasmids from different colonies were isolated and purified using QIAprep Spin Miniprep Kit (Qiagen). Plasmids were digested with BsmBI and BamHI in Cut Smart Buffer (New England BioLabs, Inc.) at 37 °C for 1 h and then analyzed by 1% agarose gel. Sequencing was performed by GENEWIZ (South Plainfield, New Jersey, NJ; Fig. S5 A–F).

Lentivirus was generated with psPAX2, pVSV-G and the pLentiCRISPR plasmids that contain the guides and Cas9 in 293T cells. Fourty-eight hours later, all supernatant was transferred to a 1.5 mL tube that was centrifuged to remove debris. The supernatant was transferred to a new 1.5 mL tube, and stored at 4 °C. HT29 cells were transfected with the lentivirus supernatant and polybrene was added to enhance the transfection. PuroMYCin (final concentration is 1 μg/mL) was added to the medium to select positive cells.

DNA was extracted and purified from positive HT29 cells using DNeasy Blood & Tissue kit (Qiagen). PCR primers that frank both sides of the exon 3 of PUMA gene were used to amplify the target region (forward, 5’-CACAGTCTCTGGCCTTCTGG-3’; reverse, 5’-AGCTGCCGCACATCTGG-3’). The amplicon is GC-rich region, to improve PCR specificity, we performed temperature gradient PCR to optimize annealing temperature. A hot-start and touch-down PCR with accuPrime Pfx DNA Polymerase (ThermoFisher Scientific) and 2.5% DMSO and 1M betaine, was performed to achieve specific amplification of target region. The PCR products were purified by QIAquick PCR purification kit (Qiagen) for Sanger sequencing. TIDE analysis was performed using an online tool (TIDE: Tracking of Indels by DEcomposition, Sequencing was performed by GENEWIZ (South Plainfield, New Jersey, NJ; Fig. S5 C).

Three hundred positive HT29 cells were placed into a 10 cm dish and incubated at 37 °C. After 2 wk, single cell colonies were selected and expanded. Western blotting using PUMA antibody was performed to screen the colonies (Fig. S5 E and F).

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