Background: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) has revolutionized the field of genome editing since its first application in human cells. Despite widespread adoption, systematic comparisons of editing efficiency across diverse human cell lines remain limited, particularly when evaluating next-generation variants including base editors and prime editors alongside conventional SpCas9.

Objective: This study aimed to systematically evaluate and compare the gene editing efficiency of five CRISPR variants-SpCas9, SaCas9, AsCpf1, BE4max, and PE3-across five human cell lines using four distinct delivery modalities.

Method: A total of 120 experimental units were analyzed using fluorescence-activated cell sorting (FACS), T7 endonuclease I (T7EI) assay, and next-generation sequencing (NGS). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test in SPSS v.27.

Key Results: SpCas9 in HEK293T cells achieved the highest on-target editing efficiency (87.3 ± 3.2%), while PE3 demonstrated the lowest off-target rate (1.9 ± 0.5%). Electroporation of ribonucleoprotein (RNP) complexes yielded the highest transfection efficiency (86.7 ± 3.8%). ANOVA revealed significant inter-variant differences (p < 0.001).