Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
Innovative Genomics Institute, University of California, Berkeley, CA, USA.
Nat Commun. 2024 Feb 26;15(1):1727. doi: 10.1038/s41467-024-45998-2.
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.
CRISPR 核糖核蛋白（RNP）在体外和体内进行基因组编辑具有重要优势，包括减少脱靶效应和免疫原性。然而，由于效率低和细胞毒性，在某些细胞类型中，RNP 的有效传递仍然具有挑战性。为了解决这些问题，我们设计了自传递的 RNP，它们可以促进有效的细胞摄取，并在不需要辅助材料或生物分子的情况下进行强大的基因组编辑。筛选与 Cas9 蛋白融合的细胞穿透肽（CPP），确定了能够有效编辑神经祖细胞基因组的有效构建体。进一步对这些融合蛋白进行工程设计，建立了 Cas9 与三个 A22p 肽的 C 端融合，A22p 肽来源于人类神经生长因子 3a，与其他构建体相比，编辑效率显著提高。我们发现，当直接注射到小鼠纹状体时，自传递 Cas9 RNP 可在临床上相关基因中产生强大的基因组编辑。总体而言，自传递 Cas9 蛋白为体外和体内基因组编辑提供了一种简便有效的平台。
