**Background:** The CRISPR/Cas system, originally discovered as an adaptive immune system in bacteria and archaea, has revolutionized genetic engineering by enabling precise, efficient, and cost-effective genome editing. This review provides a comprehensive overview of the evolution, classification, and engineering of CRISPR/Cas systems, focusing on their mechanisms, applications, and challenges. The paper covers the two main classes of CRISPR systems: class 1 (types I, III, IV) which utilize multiple Cas proteins, and class 2 (types II, V, VI) which rely on a single effector protein. The most widely used system is type II CRISPR/Cas9, which uses a single guide RNA (sgRNA) and Cas9 nuclease to generate double-strand breaks (DSBs) at target DNA sequences. Other important systems include Cas12a (type V) which recognizes T-rich PAM sequences and produces staggered DSBs, and Cas13a (type VI) which targets RNA. The review also discusses DSB repair pathways (NHEJ and HDR) and their roles in genome editing.
**Methods:** This is a narrative review that synthesizes information from the published literature on CRISPR/Cas systems. The authors describe the classification of CRISPR systems into two classes, six types, and 33 subtypes, summarizing key characteristics such as effector complexes, tracrRNA requirements, signature proteins, and target substrates. They detail the mechanisms of engineered systems including CRISPR/Cas9, Cas12a, Cas13a, base editors (CBEs and ABEs), and prime editors (PE1, PE2, PE3, PE3b, PE4, PE5). The review also covers applications in clinical trials (ex vivo and in vivo) and agriculture, as well as challenges such as off-target effects and immunogenicity.
**Key Results:** The paper reports that CRISPR/Cas9, using SpCas9 with PAM sequence 5'-NGG-3', is the most widely used system. High-fidelity variants like SpCas9-HF1, HiFiCas9, evoCas9, eSpCas9, HypaCas9, Sniper-Cas9, and xCas9 3.7 have been developed to improve specificity, though often with reduced editing efficiency. Cas12a recognizes T-rich PAM (e.g., TTTV for Acidaminococcus sp.) and generates staggered DSBs, with smaller guide RNA (~43 nt) compared to Cas9 (~101 nt). Cas13a targets ssRNA and requires a protospacer flanking site (PFS) of A, U, or C. Base editors: CBEs convert C→T with efficiencies of ~15-75% and minimal indels (≤1%); ABEs convert A→G with ~50% efficiency and very low indels (≤0.1%). Prime editing enables all types of base conversions, insertions (up to 44 bp), and deletions (up to 80 bp) with higher precision and lower off-targets than Cas9 nuclease. Clinical applications include ex vivo editing for cancer immunotherapy (PD-1 knockout in T cells), sickle cell disease (BCL11A knockout in HSPCs), HIV (CCR5 knockout), and in vivo editing for Leber's congenital amaurosis 10 (CEP290 mutation correction). Agricultural applications include drought-resistant corn, vitamin D-enriched tomatoes, heat-tolerant cattle, and double-muscled sheep.
**Clinical Implications:** The review highlights the transformative potential of CRISPR/Cas systems for treating genetic disorders, infectious diseases, and cancer. Ex vivo approaches have shown promise in clinical trials for sickle cell disease and β-thalassemia (phase 2/3), HIV (phase 1), and non-small-cell lung cancer (phase 1). In vivo editing has been used for Leber's congenital amaurosis 10. Base editors and prime editors offer safer alternatives with fewer off-target effects and the ability to correct up to 89% of known pathogenic variants. However, challenges remain, including off-target effects, immunogenicity of Cas9 proteins (especially from S. pyogenes and S. aureus), and ethical concerns regarding germline editing. The authors emphasize the need for improved delivery methods, better off-target prediction tools, and careful regulatory frameworks to ensure safe and equitable clinical translation.