Mechanism- guided engineering of a minimal biological particle for genome editing

• 成果类型: Article
• 署名作者: Ngo, Wayne; Peukes, Julia; Baldwin, Alisha; Xue, Zhiwei Wayne; Hwang, Sidney; Stickels, Robert R.; Lin, Zhi; Satpathy, Ansuman T.; Wells, James A.; Schekman, Randy; Nogales, Eva; Doudna, Jennifer A.; Bjorkman, Pamela
• 署名单位: University of California System; University of California Berkeley; University of California System; University of California San Francisco; The J David Gladstone Institutes; University of California System; University of California Berkeley; University of California System; University of California Berkeley; Stanford University; University of California System; University of California San Francisco; University of California System; University of California San Francisco; Howard Hughes Medical Institute; University of California System; University of California Berkeley; United States Department of Energy (DOE); Lawrence Berkeley National Laboratory; University of California System; University of California Berkeley
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13872
• DOI: 10.1073/pnas.2413519121
• 发表日期: 2025-01-07
• 关键词: nuclear-localization signals; hiv-1; infection; import

摘要

The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV- mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo- electron tomography and small molecule inhibitors guided the removal of similar to 80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.