Abstract

Type V CRISPR-Cas12a systems are an attractive alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells. Here we describe optimization to the nuclear localization signal composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in mammalian cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. A 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not decrease the specificity of editing in HEK293T and CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.