Chen, K.; Stahl, E. C.; Kang, M. H.; Xu, B.; Allen, R.; Trinidad, M.; Doudna, J. A.

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects1. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineered self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identified potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins identified a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibited substantially improved editing efficacy compared to other constructs. We found that self-deliverable Cas9 RNPs generated robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.