Assess graduate-level understanding of the molecular mechanisms by which CRISPR-Cas9 recognizes and cleaves DNA—including PAM recognition, guide RNA–DNA base pairing, seed-region and R-loop formation, conformational changes and cleavage chemistry—and the molecular causes of off-target activity such as mismatch tolerance, bulges, chromatin context, DNA accessibility, reaction kinetics, and nuclease/guide design; include analysis of how experimental variables and engineering strategies (high-fidelity variants, truncated guides, paired nickases, altered PAM specificity) affect specificity and prediction of off-targets.