From Double-Strand Breaks to Precision Edits: A Comparative Review of Modern Genome Editing Tools

Abstract

Over the past decade, genome editing has been transformed by RNA‑guided CRISPR‑Cas systems, which have enabled increasingly precise and programmable DNA modification. Since CRISPR‑Cas9 was repurposed for genome editing in 2012, base editing (2016) and prime editing (2019) have expanded the field beyond double‑strand break–dependent repair. Two classes of DNA base-editors have been developed, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime editing has further expanded the CRISPR editing toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Base editors enable precise transition mutations without double-strand breaks (DSBs). In HEK293T cells, CBE3 has demonstrated a 2‑ to 6‑fold improvement over CBE2 while maintaining low indel formation (~1.1%). Advances in ABE development include seventh‑generation variants achieving ~50% efficiency with ≥99.9% product purity and minimal indels in human cells. In HEK293T cells, third-generation prime editors (PE3) achieved editing efficiencies of up to ~55%, while the subsequent PE3b strategy reduced indel formation by up to 13-fold without compromising editing efficiency. CRISPR-Cas9 has achieved clinical success in the treatment of sickle cell disease and β-thalassemia, while base editing and prime editing have shown promising preclinical potential for disorders such as cystic fibrosis and Tay-Sachs disease. Together, these findings highlight the rapid progress of genome editing while underscoring remaining challenges in delivery, specificity, and clinical translation. This review compares Cas9-mediated homology-directed repair (HDR) with generations of cytosine base editors (CBE1–CBE3), adenine base editors (ABE1-ABE7), and prime editors (PE1–PE3b), focusing on their mechanistic distinctions, efficiencies, delivery challenges, and therapeutic applications.