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Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR - Nat. Biotechnol. (2014)

Bottom Line: Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
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.

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Delivery of Cre recombinase to cultured human cells. (a) Fusion of either highly cationic (+36)GFP or highly anionic (−30)GFP to Cre recombinase. We used a HeLa reporter cell line that expresses DsRed upon Cre-mediated recombination to evaluate Cre delivery efficiency. (b) HeLa dsRed cells treated with 10 nM (−30)GFP-Cre and 1.5 µL of the cationic lipid formulation RNAiMAX. Cells were visualized after incubation for 48 hours in media containing 10% fetal bovine serum (FBS). (c) Delivery of (+36)GFP-Cre in 10% FBS media or in serum-free media, and (−30)GFP-Cre with or without the cationic lipid RNAiMAX (0.8 µL) in full-serum media. (d) Effect of cationic lipid dose on functional (−30)GFP-Cre delivery efficacy after 48 hours. (e) Comparison of several commercially available cationic lipids and polymers for functional delivery efficacy of (−30)dGFP-Cre. (f) RNAiMAX-mediated delivery of multiple anionic peptide or protein sequences fused to Cre. The net theoretical charge of the VP64 activation domain and the 3xFLAG tag is −22 and −7, respectively. All experiments were performed with 25 nM protein in 48-well plate format using 275 µL DMEM with 10% FBS and no antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.
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Figure 2: Delivery of Cre recombinase to cultured human cells. (a) Fusion of either highly cationic (+36)GFP or highly anionic (−30)GFP to Cre recombinase. We used a HeLa reporter cell line that expresses DsRed upon Cre-mediated recombination to evaluate Cre delivery efficiency. (b) HeLa dsRed cells treated with 10 nM (−30)GFP-Cre and 1.5 µL of the cationic lipid formulation RNAiMAX. Cells were visualized after incubation for 48 hours in media containing 10% fetal bovine serum (FBS). (c) Delivery of (+36)GFP-Cre in 10% FBS media or in serum-free media, and (−30)GFP-Cre with or without the cationic lipid RNAiMAX (0.8 µL) in full-serum media. (d) Effect of cationic lipid dose on functional (−30)GFP-Cre delivery efficacy after 48 hours. (e) Comparison of several commercially available cationic lipids and polymers for functional delivery efficacy of (−30)dGFP-Cre. (f) RNAiMAX-mediated delivery of multiple anionic peptide or protein sequences fused to Cre. The net theoretical charge of the VP64 activation domain and the 3xFLAG tag is −22 and −7, respectively. All experiments were performed with 25 nM protein in 48-well plate format using 275 µL DMEM with 10% FBS and no antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.

Mentions: First, we tested whether the engineered supernegatively charged GFP variant,28 (−30)GFP, could mediate complexation and delivery of fused protein cargo (Fig. 1b). We translationally fused (−30)GFP to Cre recombinase to generate (−30)GFP-Cre; note that (−30) refers to the net theoretical charge of the GFP moiety, not the net charge of the fusion. We assayed a variety of commercially available cationic lipids for their ability to functionally deliver (−30)GFP-Cre into HeLa cells that only express DsRed upon Cre-mediated recombination (Fig. 2a). Lipofectamine RNAiMAX (Life Technologies), hereafter referred to as “RNAiMAX”, is a commercial reagent designed for delivery of siRNAs. Delivery of 10 nM (−30)GFP-Cre complexed with 1.5 µL RNAiMAX in DMEM (Dulbecco’s Modified Eagle’s Media plus GlutaMAX, Life Technologies) containing 10% fetal bovine serum (FBS) led to strong DsRed fluorescence signal among treated cells. Flow cytometry revealed that 48 hours after treatment, 52% of cells expressed DsRed consistent with Cre recombination (Fig. 2b).


Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR - Nat. Biotechnol. (2014)

Delivery of Cre recombinase to cultured human cells. (a) Fusion of either highly cationic (+36)GFP or highly anionic (−30)GFP to Cre recombinase. We used a HeLa reporter cell line that expresses DsRed upon Cre-mediated recombination to evaluate Cre delivery efficiency. (b) HeLa dsRed cells treated with 10 nM (−30)GFP-Cre and 1.5 µL of the cationic lipid formulation RNAiMAX. Cells were visualized after incubation for 48 hours in media containing 10% fetal bovine serum (FBS). (c) Delivery of (+36)GFP-Cre in 10% FBS media or in serum-free media, and (−30)GFP-Cre with or without the cationic lipid RNAiMAX (0.8 µL) in full-serum media. (d) Effect of cationic lipid dose on functional (−30)GFP-Cre delivery efficacy after 48 hours. (e) Comparison of several commercially available cationic lipids and polymers for functional delivery efficacy of (−30)dGFP-Cre. (f) RNAiMAX-mediated delivery of multiple anionic peptide or protein sequences fused to Cre. The net theoretical charge of the VP64 activation domain and the 3xFLAG tag is −22 and −7, respectively. All experiments were performed with 25 nM protein in 48-well plate format using 275 µL DMEM with 10% FBS and no antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.
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Related In: Results  -  Collection

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Figure 2: Delivery of Cre recombinase to cultured human cells. (a) Fusion of either highly cationic (+36)GFP or highly anionic (−30)GFP to Cre recombinase. We used a HeLa reporter cell line that expresses DsRed upon Cre-mediated recombination to evaluate Cre delivery efficiency. (b) HeLa dsRed cells treated with 10 nM (−30)GFP-Cre and 1.5 µL of the cationic lipid formulation RNAiMAX. Cells were visualized after incubation for 48 hours in media containing 10% fetal bovine serum (FBS). (c) Delivery of (+36)GFP-Cre in 10% FBS media or in serum-free media, and (−30)GFP-Cre with or without the cationic lipid RNAiMAX (0.8 µL) in full-serum media. (d) Effect of cationic lipid dose on functional (−30)GFP-Cre delivery efficacy after 48 hours. (e) Comparison of several commercially available cationic lipids and polymers for functional delivery efficacy of (−30)dGFP-Cre. (f) RNAiMAX-mediated delivery of multiple anionic peptide or protein sequences fused to Cre. The net theoretical charge of the VP64 activation domain and the 3xFLAG tag is −22 and −7, respectively. All experiments were performed with 25 nM protein in 48-well plate format using 275 µL DMEM with 10% FBS and no antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.
Mentions: First, we tested whether the engineered supernegatively charged GFP variant,28 (−30)GFP, could mediate complexation and delivery of fused protein cargo (Fig. 1b). We translationally fused (−30)GFP to Cre recombinase to generate (−30)GFP-Cre; note that (−30) refers to the net theoretical charge of the GFP moiety, not the net charge of the fusion. We assayed a variety of commercially available cationic lipids for their ability to functionally deliver (−30)GFP-Cre into HeLa cells that only express DsRed upon Cre-mediated recombination (Fig. 2a). Lipofectamine RNAiMAX (Life Technologies), hereafter referred to as “RNAiMAX”, is a commercial reagent designed for delivery of siRNAs. Delivery of 10 nM (−30)GFP-Cre complexed with 1.5 µL RNAiMAX in DMEM (Dulbecco’s Modified Eagle’s Media plus GlutaMAX, Life Technologies) containing 10% fetal bovine serum (FBS) led to strong DsRed fluorescence signal among treated cells. Flow cytometry revealed that 48 hours after treatment, 52% of cells expressed DsRed consistent with Cre recombination (Fig. 2b).

Bottom Line: Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
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.

Show MeSH