Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing

• Published in: Nature biotechnology Vol. 35; no. 12; pp. 1179 - 1187
• Main Authors: Yin, Hao, Song, Chun-Qing, Suresh, Sneha, Wu, Qiongqiong, Walsh, Stephen, Rhym, Luke Hyunsik, Mintzer, Esther, Bolukbasi, Mehmet Fatih, Zhu, Lihua Julie, Kauffman, Kevin, Mou, Haiwei, Oberholzer, Alicia, Ding, Junmei, Kwan, Suet-Yan, Bogorad, Roman L, Zatsepin, Timofei, Koteliansky, Victor, Wolfe, Scot A, Xue, Wen, Langer, Robert, Anderson, Daniel G
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.12.2017; Nature Publishing Group
• Subjects: 101/62; 38/77; 45/23; 45/41; 631/1647/1511; 631/61/391; 64/60; 96/106; 96/109; 96/31; Agriculture; Animals; Biochemical genetics; Bioinformatics; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Chemical modification; Cholesterol; CRISPR-Cas Systems - genetics; Editing; Formulations; Gene Editing - methods; Gene targeting; Gene Transfer Techniques; Genome editing; Genomes; Intravenous administration; Life Sciences; Liver; Liver - metabolism; Methods; Mice; mRNA; Nanoparticles; Nanoparticles - chemistry; Nuclease; Nucleic Acid Conformation; Proprotein Convertase 9 - genetics; Ribonucleic acid; RNA; RNA modification; RNA processing; RNA, Guide, CRISPR-Cas Systems; Therapeutic applications
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt.4005

Efficient Cas9 genome editing in vivo is achieved without viral vectors using chemically modified single-guide RNAs. Efficient genome editing with Cas9–sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9–sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications. Using lipid nanoparticle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single intravenous injection into mice induces >80% editing of Pcsk9 in the liver. Serum Pcsk9 is reduced to undetectable levels, and cholesterol levels are significantly lowered about 35% to 40% in animals. This strategy may enable non-viral, Cas9-based genome editing in the liver in clinical settings.