Engineering self-deliverable ribonucleoproteins for genome editing in the brain

Kai Chen, Elizabeth C. Stahl, Min Hyung Kang, Bryant Xu, Ryan Allen, Marena Trinidad and Jennifer A. Doudna ()
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Kai Chen: University of California, Berkeley
Elizabeth C. Stahl: University of California, Berkeley
Min Hyung Kang: University of California, Berkeley
Bryant Xu: University of California, Berkeley
Ryan Allen: University of California, Berkeley
Marena Trinidad: University of California, Berkeley
Jennifer A. Doudna: University of California, Berkeley

Nature Communications, 2024, vol. 15, issue 1, 1-11

Abstract: 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 effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer 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 identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate 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.

Date: 2024
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DOI: 10.1038/s41467-024-45998-2

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