Beyond CRISPR: next-gen precision engineering of CAR-NK cells for enhanced persistence, trafficking, and tumor eradication

Chimeric antigen receptor natural killer (CAR-NK) cells represent a promising "off-the-shelf" alternative to CAR-T cells, offering a superior safety profile and inherent multi-antigen targeting capabilities. However, their clinical potential is constrained by the "CRISPR ceiling", a set of practical limitations of DSB-based CRISPR-Cas9 such as DNA double-strand break (DSB)-associated chromosomal rearrangements and p53-mediated fitness loss, low efficiency for safe, large, multicistronic knock-ins, and rigid promoter-driven transgene expression that can cause tonic signaling. Importantly, next-generation, DSB-free base and prime editors reduce or eliminate the DSB-associated genotoxic stress observed with nuclease cutting, CRISPR-associated transposases now enable programmable, targeted insertion strategies that can accommodate larger cassettes, and synthetic/epigenetic circuits provide dynamic, context-dependent transgene control that avoids constitutive promoter-driven tonic signaling. This review explores technological approaches beyond conventional CRISPR, highlighting next-generation precision engineering tools that may enable improved CAR-NK therapies and represent potential advances in safety and efficacy. We detail how base editing, epigenetic reprogramming, targeted transposon systems, and synthetic biology circuits can be synergistically integrated to overcome critical clinical challenges. These advanced technologies enable the precise enhancement of three fundamental pillars of efficacy: Persistence through endogenous cytokine armoring and metabolic engineering; Trafficking via chemokine receptor matching and stromal barrier degradation; and Tumor Eradication using logic-gated targeting, immunomodulatory payloads, and bispecific engagers. By synthesizing these cutting-edge advances, we provide a roadmap for developing next-generation CAR-NK cells capable of durable, potent, and safe antitumor responses against both hematological and solid malignancies, ultimately forging a new frontier in accessible cellular immunotherapy.