**Background:** The CRISPR-Cas9 system has revolutionized genome editing by enabling targeted modifications. Wild-type Cas9 induces double-strand breaks (DSBs) that are repaired by error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR). Engineered Cas9 variants, such as base editors (BEs) and prime editors (PEs), allow precise single-base changes without DSBs. This review focuses on the mechanisms, evolution, and therapeutic applications of these tools.
**Methods:** The authors conducted a narrative review of the literature, covering the discovery and engineering of Cas9 nucleases, base editors, prime editors, CRISPR-associated transposon systems, and delivery methods. They summarized preclinical and clinical studies using these tools for gene disruption, correction, and regulation.
**Key Results:**
- Cas9 nucleases can disrupt genes via indels (e.g., targeting VEGFR2 for angiogenesis, TTR for amyloidosis, PCSK9 for hypercholesterolemia). Dual sgRNA strategies enable exon skipping (e.g., DMD gene) or deletion of mutant fragments (e.g., CEP290 IVS26 for LCA10).
- HDR-mediated gene correction has been achieved in mouse models for OTC deficiency (10% hepatocyte correction), PKU (partial PAH activity restoration), and LDLR mutation (reduced cholesterol).
- Base editors (CBEs and ABEs) efficiently convert C·G to T·A or A·T to G·C. In vivo, CBE targeting Pcsk9 reduced plasma PCSK9 and cholesterol in mice. ABE corrected PAH mutation in PKU mice, normalizing phenylalanine levels. eTAM (CBE) achieved >50% exon skipping in DMD, restoring up to 90% dystrophin in heart.
- Prime editors can install all 12 point mutations and small indels. Optimization (e.g., paired pegRNAs, MMR inhibition via dominant-negative MLH1) improved efficiency. PE4/PE5 variants reduced indels.
- Delivery: AAV (limited by ~4.7 kb capacity) uses split Cas9 or compact orthologs (SaCas9, 3.2 kb). LNP delivered Cas9 mRNA/sgRNA achieved 80% Pcsk9 editing in mice and 73% TTR editing in monkeys, with serum TTR reduction >94%. eVLPs achieved 63% Pcsk9 editing in mouse liver and 60% base editing in brain.
- Clinical trials: >34 trials initiated. For example, CTX001 (BCL11A enhancer disruption) reduced transfusion needs in β-thalassemia and vaso-occlusive episodes in sickle cell disease. EDIT-101 (CEP290 deletion) is in Phase 1/2 for LCA10. LNP-delivered Cas9 targeting TTR reduced serum TTR by up to 87% in patients.
**Clinical Implications:** CRISPR-Cas9 tools offer transformative potential for treating genetic diseases, metabolic disorders, and cancer. Base editors and prime editors provide precise, irreversible edits with reduced off-target effects compared to nuclease-based approaches. However, challenges remain: off-target editing (sequence-dependent and independent), pre-existing immunity to Cas9 (prevalent in ~50% of humans), and delivery to non-liver tissues. Strategies to mitigate these include high-fidelity Cas9 variants, transient expression (LNP vs. AAV), and engineered delivery systems (SORT LNPs, eVLPs). The review emphasizes that single-dose therapies are preferred to avoid immune responses. Ongoing clinical trials will determine long-term safety and efficacy.