Triplicate transfections on three separate days. Conditions: (1) BE4max + STOP sgRNA, (2) BE4max + non-targeting sgRNA, (3) BE4max-dead-deaminase (catalytically inactive) + STOP sgRNA, (4) GFP-targeting Cas9 nuclease + same-locus sgRNA (DSB comparator), (5) mock. EGFP-loss read at day 5 by flow cytometry (≥10,000 events). Genomic DNA at day 5 for amplicon-seq to measure C-to-T, bystander, and indel rates. 2 x 10^5 cells per transfection.
BSL-2 for HEK293T and for any lentiviral generation of the EGFP-stable line (the stable line itself, once made and tested, is handled at BSL-2). Class II biosafety cabinet for culture. Lipofectamine and plasmid DNA are low-hazard. DAPI/7-AAD are potential mutagens — wear gloves and dispose appropriately. Decontaminate cultureware with 10% bleach. Standard PPE: gloves, lab coat, eye protection. No DSB-associated radiation or special hazard.
Non-targeting sgRNA + BE4max controls for editor-independent fluorescence drift and transfection toxicity. Dead-deaminase BE4max + STOP sgRNA isolates deaminase activity from sgRNA/nickase effects (should show no C-to-T, no EGFP loss). Cas9 nuclease at the same locus is the DSB comparator showing indel-mediated knockout and higher indel rate. Mock (lipid only) sets baseline EGFP+ percentage. Untransfected EGFP-stable cells define the EGFP+ gate; parental EGFP-negative HEK293T set the EGFP- gate.
Active BE4max + STOP guide: >50% target C-to-T conversion, >40% EGFP-negative, indels <2%, bystander editing dependent on motif (ideally low). Non-targeting and dead-deaminase controls: EGFP-negative <3%, C-to-T <1%. Cas9 comparator: high indels (>60%) with EGFP loss but a different mutational signature. Viability >80% across base-editor conditions (lower for nuclease). EGFP-loss should correlate with C-to-T conversion across replicates.
Inactivate a stably expressed EGFP reporter in HEK293T cells without inducing double-strand breaks by using a cytidine deaminase base editor (BE4max) and an sgRNA positioning a target C within the editing window to convert a glutamine/arginine codon to a stop codon, quantifying inactivation by EGFP-negative percentage and the underlying C-to-T conversion by amplicon sequencing.
Independent variable: editor/sgRNA combination (active BE + STOP guide vs controls). Dependent variables: % EGFP-negative cells, target C-to-T conversion (%), bystander C-to-T at adjacent cytosines (%), indel frequency (%), cell viability (%). Controlled variables: cell number (2 x 10^5), plasmid amounts (500 ng editor, 167 ng sgRNA, 3:1 ratio), Lipofectamine volume, transfection-to-readout time (day 5), flow cytometer voltages/gating, amplicon and sequencing depth.
Co-delivery of BE4max and a CRISPR-STOP sgRNA placing the target cytosine at positions 4-8 of the protospacer will yield >50% C-to-T conversion at the target site and a corresponding >40% EGFP-negative population, with minimal indels (<2%) and acceptable bystander editing, demonstrating efficient DSB-free knockout.
Flow cytometry analyzed in FlowJo: gate singlets/live, quantify %EGFP-negative relative to mock; export median EGFP fluorescence. Amplicon-seq processed in CRISPResso2 base-editor mode reporting per-position nucleotide conversion frequencies, distinguishing target vs bystander cytosines and total indels. Correlate %EGFP-negative with target C-to-T across replicates (Pearson). Normalize EGFP-loss to the non-targeting control to subtract editor-independent drift.
Low C-to-T conversion: target C outside the optimal editing window (positions 4-8) — redesign the sgRNA to reposition the C, or use a YE1/evolved BE variant. High indels with BE4max: nickase generating breaks at high editor dose — reduce plasmid and confirm BE4max (not Cas9) identity. EGFP loss without C-to-T (false phenotype): silencing or toxicity — check the dead-deaminase control and viability dye. Excessive bystander editing: multiple Cs in window — switch to a narrowed-window editor or accept if non-coding. Poor transfection efficiency: cells over-confluent or DNA quality low — reseed at 60-70%, use endotoxin-free maxiprep DNA.
Compare %EGFP-negative and target C-to-T across the five conditions by one-way ANOVA (n = 3) with Dunnett's correction versus the non-targeting control, alpha = 0.05. Correlate conversion vs phenotype by Pearson r with two-tailed p. With n = 3 and the expected large difference between active and dead editor (>10 SD), power exceeds 0.95. Report bystander/indel rates as mean +/- SD; no additional correction needed beyond the ANOVA post-hoc adjustment.