Expression and purification of cell-penetrating Cas9 and Cas12a enzymes for peptide-assisted genome editing.

Autor:	Cuomo RG; Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States., Zhang Z; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, United States; Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, United States., Yamada K; Bioengineering Graduate Group, University of Pennsylvania, Philadelphia, PA, United States., Krosky AJ; Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States., Shi J; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, United States; Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, United States; Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: jushi@upenn.edu., Kohli RM; Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, United States; Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: rkohli@pennmedicine.upenn.edu., Parker JB; Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2024; Vol. 705, pp. 25-49. Date of Electronic Publication: 2024 Sep 05.
DOI:	10.1016/bs.mie.2024.07.009
Abstrakt:	Recent advances in CRISPR-Cas genomic editors have shifted us ever closer to achieving the ultimate therapeutic goal of accomplishing any edit in any cell. However, delivery of this editing machinery to primary cells with high efficiency while avoiding cellular toxicity remains a formidable challenge. Peptide-Assisted Genome Editing (PAGE) provides a simple, modular, and rapid approach for the protein-based delivery of CRISPR-Cas proteins or ribonucleoprotein complexes into primary cells with high efficiency and minimal cytotoxicity. In this chapter, we detail an expression and purification protocol to obtain highly pure Cas9-T6N and opCas12a-T8N PAGE genomic editors. The robustness of this protocol allows for consistent preparations of the purified editors that can be reliably used for the editing of primary and immortalized cells. (Copyright © 2024. Published by Elsevier Inc.)
Databáze:	MEDLINE
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