Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo
By John A. Zuris, David B. Thompson, Y. Shu, J. Guilinger, Jeffrey L Bessen, J. H. Hu, Morgan L. Maeder, J. K. Joung, Zheng-Yi Chen, David R Liu and 1 more. Nature Biotechnology, 2014
1,242 citations recorded by the source corpus.
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.
Subfields: Molecular Biology
View Paper: https://www.semanticscholar.org/paper/c1c58a7157d797b5e177624e14ea8a95ac6d0291
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In Molecular Biology
- Jennifer A. Doudna
- Haifeng Wang
- Marie La Russa
- Lei S. Qi
- Steven Lin
- Jerilyn A. Timlin
- J. Carney
- J. C. Harper
Citations in the corpus
- Engineering the Delivery System for CRISPR-based Genome Editing (2018, background): “The commercial lipid transfection reagent Lipofectamine is a well-established delivery vehicle for nucleic acids and gene editing proteins [20, 43].”
- CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “This method allowed for gene modification of up to 80% in cultured cells and approximately 20% in mouse inner ear hair cells in vivo (257).”
- Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA[OPEN] (2015, background): “In mammalian systems, both Cas9 and gRNA can be delivered transiently in the form of RNA-protein complexes providing high mutation frequencies (Kim et al., 2014; Lin et al., 2014; Zuris et al., 2015).”