Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery

Thomas Gaj, Brett T. Staahl, Goncalo M.C. Rodrigues, Prajit Limsirichai, Freja K. Ekman, Jennifer A. Doudna, David V. Schaffer

Research output: Contribution to journalArticle

Abstract

Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the highfidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNAoligonucleotide. However, combining RNPswith transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homologydirected genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing.

Original languageEnglish (US)
Article numbere98
JournalNucleic acids research
Volume45
Issue number11
DOIs
StatePublished - Jun 1 2017
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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    Gaj, T., Staahl, B. T., Rodrigues, G. M. C., Limsirichai, P., Ekman, F. K., Doudna, J. A., & Schaffer, D. V. (2017). Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery. Nucleic acids research, 45(11), [e98]. https://doi.org/10.1093/nar/gkx154