An immune-inspired spatiotemporal switch for precise CRISPR genome editing with reduced off-target effects and chromosomal translocations

Off-target mutations and chromosomal translocations are a fundamental obstacle to precise CRISPR-Cas9 genome editing—a challenge strikingly analogous to that faced by activation-induced cytidine deaminase (AID), which must introduce targeted DNA deamination at the immunoglobulin heavy chain (IgH) locus for antibody diversification while avoiding genome-wide collateral damage. In B cells, AID has evolved a C-terminal nuclear export signal (NES) that constitutively sequesters it in the cytoplasm, restricting genomic access to the brief mitotic window when the nuclear envelope breaks down.

This spatiotemporal confinement ensures efficient on-target editing while strictly limiting off-target deamination across the genome. Inspired by this evolutionarily refined immune regulatory mechanism, we engineered a Cas9 variant fused to AID’s NES, confining its nuclear access to mitosis. This confinement preserves on-target editing efficiency while dramatically reducing off-target mutations and chromosomal translocations.