CasPER reduces mutagenic repair during targeted transgene integration in human immune cells

Abstract Efficient targeted integration is not necessarily safe: CRISPR–Cas9-induced DNA double-strand breaks can generate indels and chromosomal rearrangements even with highly specific gRNAs. Here, we developed CasPER (Cas9–PIAS4 for error reduction), a genome-editing platform that localises the SUMO E3 ligase PIAS4 to Cas9-induced breaks to modulate repair. Across multiple loci, cargo sizes, cell types and donos, CasPER maintained targeted transgene integration while reducing mutagenic indels and improving editing precision. The platform was compatible with plasmid-, mRNA- and RNP-based editor delivery and with linear DNA, circular DNA and AAV donor templates. In primary human NK cells, CasPER achieved knock-in comparable to Cas9 but generated fewer on-target indels. CAST-seq further showed that structural rearrangements can arise despite the absence of detectable off-target indels and that CasPER was associated with fewer distal rearrangement events. In primary human T cells, CasPER enabled targeted CAR insertion and generated functional CAR-T cells exhibiting tumour-cell killing, cytokine secretion and activation. Together, these findings distinguish cleavage specificity from repair fidelity and establish CasPER as a versatile platform for reducing genome-editing-associated mutagenesis while preserving therapeutically relevant cell function.