CRISPR-associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons, resulting in KRAB(KOX1)-specific degradation.

The use of CRISPR-associated enzymes in induced pluripotent stem cell (iPSC)-derived neurons presents unique challenges compared with dividing cell lines. For example, loss of dCas9-KRAB expression after differentiation has been observed and largely ascribed to transgene silencing. Here, we investigated the expression of CRISPR enzymes in iPSCs and Ngn2-derived neurons. We found that the commonly used dCas9-KRAB(KOX1) displayed a dramatic reduction in protein levels following differentiation, yet nCas9 constructs retained comparable levels. We further found that CRISPR constructs, primarily relying on the SV40 nuclear localization signal (NLS), fail to localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs, leading to KRAB(KOX1)-specific cytoplasmic degradation. By testing other NLSs, we rescued neuronal nuclear localization and protein expression, confirming the contribution of mislocalization to the instability of dCas9-KRAB(KOX1) in neurons. As the lack of nuclear localization can have a profound impact on CRISPR activity, we suggest further investigation across cultured and in vivo postmitotic cell models. A record of this paper's transparent peer review process is included in the supplemental information.