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bioRxiv · 10.1101/2023.11.15.567251

Engineering self-deliverable ribonucleoproteins for genome editing in the brain

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

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.

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Chen, K., Stahl, E. C., Kang, M. H., Xu, B., Allen, R., Trinidad, M., Doudna, J. A.. 2023-11-15. Engineering self-deliverable ribonucleoproteins for genome editing in the brain. https://doi.org/10.1101/2023.11.15.567251

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