Q5 hot start high fidelity 2x master mix polymerase

Fluorescence-activated cell sorting was performed 72 h after transfection using MoFlo Astrios. The cells were collected after washing with PBS and digesting with 0.25% trypsin, then centrifuged at 200 g for 3 min and resuspended in PBS. For split PE, mCherry and EGFP double-positive cells were sorted, whereas for PE2, EGFP-positive cells were sorted, indicating the successful transfection of Cas9 and MLV expression vectors into the sorted cells. Approximately 10,000-50,000 sorted cells were subjected to further analysis. The sorted cells were lysed with NP40 following the condition: 56 °C for 60 min and 96 °C for 10 min. Subsequently, PCR amplification of the target site was performed using Q5 Hot Start High-Fidelity 2X Master Mix polymerase (NEB, M0494S).

EXO editor constructs were designed based on the NCBI Protein Exonuclease 1 (accession Q9UQ84) and NCBI Nucleotide Homo sapiens exonuclease 1 transcript variant 4 mRNA (accession NM_001319224.2). Sequences for amino acids 1–352 of exonuclease 1, E4, E1B, and DN1S were synthesized by Guangzhou IGE Biotechnology. All of them were separately cloned into Cas9 expression vector backbone (Addgene, #41815). Guide RNA sequences were cloned into the BpiI-digested backbone (Addgene, #48962) and designed through CRISPR RGEN Tools (http://www.rgenome.net). dsDNA donors use pFlexibleDT as the backbone. The insertion sequences were located between PmeI and NotI sites and amplified by PCR using Q5 Hot Start High-Fidelity 2X Master Mix polymerase (NEB, M0494S). For knock-in at hRosa26, hAAVS1, and hGAPDH loci, donors with about 1000 bp homology arms were amplified from human genome and plasmid (SA-EGFP for hRosa26 and hAAVS1 loci, T2A-mCherry for hGAPDH, hTBP, and hRPL5 loci, T2A-EGFP for hACTB and hH2BC12 loci). The PCR products were gel-purified using HiPure Gel Pure DNA Mini Kit (Magen, D2111-03). The purified PCR products were integrated into linearized backbone using pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, CU101-02).