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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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DNA sequence specificity of Cas9-mediated endogenous gene cleavage in cultured human cells by plasmid transfection or by cationic lipid-mediated protein:sgRNA delivery using 1.6 µL RNAiMAX complexed with 100 nM Cas9 and 100 nM sgRNA. (a) T7EI assay was performed for on-target modification of endogenous CLTA, EMX, and VEGF genes in HEK293T cells. (b–d) On-target:off-target DNA modification ratio resulting from Cas9:sgRNA for plasmid transfection or cationic lipid-mediated protein:sgRNA delivery. The conditions for each treatment were adjusted to result in ~10% on-target cleavage, enabling a comparison of DNA cleavage specificity between the two delivery methods under conditions in which on-target gene modification efficiencies are similar. P values for a single biological replicate are listed in Supplementary Table 2. Each on- and off-target sample was sequenced once with > 10,000 sequences analyzed per on-target sample and an average of > 111,000 sequences analyzed per off-target sample (Supplementary Table 2). All protein:sgRNA deliveries and plasmid transfections were performed in 24-well format using 1.6 µL RNAiMAX in 550 µL DMEM-FBS without antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.
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Figure 5: DNA sequence specificity of Cas9-mediated endogenous gene cleavage in cultured human cells by plasmid transfection or by cationic lipid-mediated protein:sgRNA delivery using 1.6 µL RNAiMAX complexed with 100 nM Cas9 and 100 nM sgRNA. (a) T7EI assay was performed for on-target modification of endogenous CLTA, EMX, and VEGF genes in HEK293T cells. (b–d) On-target:off-target DNA modification ratio resulting from Cas9:sgRNA for plasmid transfection or cationic lipid-mediated protein:sgRNA delivery. The conditions for each treatment were adjusted to result in ~10% on-target cleavage, enabling a comparison of DNA cleavage specificity between the two delivery methods under conditions in which on-target gene modification efficiencies are similar. P values for a single biological replicate are listed in Supplementary Table 2. Each on- and off-target sample was sequenced once with > 10,000 sequences analyzed per on-target sample and an average of > 111,000 sequences analyzed per off-target sample (Supplementary Table 2). All protein:sgRNA deliveries and plasmid transfections were performed in 24-well format using 1.6 µL RNAiMAX in 550 µL DMEM-FBS without antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.

Mentions: Transient delivery of functional Cas9:sgRNA protein:RNA complexes circumvents risks associated with viral or other gene delivery methods and has the potential to improve the specificity of genome editing by minimizing the opportunity of agents to modify off-target substrates after the target locus is modified, or to reverse on-target modification. To test whether our approach can disrupt endogenous genes in human cells, we targeted genomic loci in the EMX1, CLTA2, and VEGF genes due to their potential biomedical relevance and their use in previous studies32,40,41 of Cas9 off-target cleavage activity. Cationic lipid-mediated delivery of Cas9:sgRNA complexes into HEK293T cells resulted in robust cleavage of all three human genes with efficiencies similar to or greater than those of plasmid transfection methods as revealed by the T7EI assay (Fig. 5a).


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)

DNA sequence specificity of Cas9-mediated endogenous gene cleavage in cultured human cells by plasmid transfection or by cationic lipid-mediated protein:sgRNA delivery using 1.6 µL RNAiMAX complexed with 100 nM Cas9 and 100 nM sgRNA. (a) T7EI assay was performed for on-target modification of endogenous CLTA, EMX, and VEGF genes in HEK293T cells. (b–d) On-target:off-target DNA modification ratio resulting from Cas9:sgRNA for plasmid transfection or cationic lipid-mediated protein:sgRNA delivery. The conditions for each treatment were adjusted to result in ~10% on-target cleavage, enabling a comparison of DNA cleavage specificity between the two delivery methods under conditions in which on-target gene modification efficiencies are similar. P values for a single biological replicate are listed in Supplementary Table 2. Each on- and off-target sample was sequenced once with > 10,000 sequences analyzed per on-target sample and an average of > 111,000 sequences analyzed per off-target sample (Supplementary Table 2). All protein:sgRNA deliveries and plasmid transfections were performed in 24-well format using 1.6 µL RNAiMAX in 550 µL DMEM-FBS without 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 5: DNA sequence specificity of Cas9-mediated endogenous gene cleavage in cultured human cells by plasmid transfection or by cationic lipid-mediated protein:sgRNA delivery using 1.6 µL RNAiMAX complexed with 100 nM Cas9 and 100 nM sgRNA. (a) T7EI assay was performed for on-target modification of endogenous CLTA, EMX, and VEGF genes in HEK293T cells. (b–d) On-target:off-target DNA modification ratio resulting from Cas9:sgRNA for plasmid transfection or cationic lipid-mediated protein:sgRNA delivery. The conditions for each treatment were adjusted to result in ~10% on-target cleavage, enabling a comparison of DNA cleavage specificity between the two delivery methods under conditions in which on-target gene modification efficiencies are similar. P values for a single biological replicate are listed in Supplementary Table 2. Each on- and off-target sample was sequenced once with > 10,000 sequences analyzed per on-target sample and an average of > 111,000 sequences analyzed per off-target sample (Supplementary Table 2). All protein:sgRNA deliveries and plasmid transfections were performed in 24-well format using 1.6 µL RNAiMAX in 550 µL DMEM-FBS without antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.
Mentions: Transient delivery of functional Cas9:sgRNA protein:RNA complexes circumvents risks associated with viral or other gene delivery methods and has the potential to improve the specificity of genome editing by minimizing the opportunity of agents to modify off-target substrates after the target locus is modified, or to reverse on-target modification. To test whether our approach can disrupt endogenous genes in human cells, we targeted genomic loci in the EMX1, CLTA2, and VEGF genes due to their potential biomedical relevance and their use in previous studies32,40,41 of Cas9 off-target cleavage activity. Cationic lipid-mediated delivery of Cas9:sgRNA complexes into HEK293T cells resulted in robust cleavage of all three human genes with efficiencies similar to or greater than those of plasmid transfection methods as revealed by the T7EI assay (Fig. 5a).

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