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CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation.

Han X, Liu Z, Jo MC, Zhang K, Li Y, Zeng Z, Li N, Zu Y, Qin L - Sci Adv (2015)

Bottom Line: It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9.This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium.We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA. ; Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065, USA.

ABSTRACT
The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9-mediated knockin system.

No MeSH data available.


Related in: MedlinePlus

Microfluidic platform for cell phenotype and gene function analysis.(A) MCF7 cells delivered with plasmids encoding only Cas9 protein or both sgPten and Cas9 protein were cultured for 7 days and then analyzed by Western blotting with the indicated antibodies. Actin was used as a loading control. The symbol * indicates long exposure. (B) Cells (5 × 104) from (A) were seeded in 60-mm dishes in complete medium and cultured for 7 days. Cells were trypsinized and collected for cell count in a Countess II FL Automated Cell Counter (Life Technologies) daily for 7 days. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (C) HeLa cells delivered with plasmids encoding only Cas9 protein or both sg53BP1 and Cas9 protein were cultured 7 days. Then, the cells were treated with 1 μM CPT for 2 hours and then examined by immunostaining with anti-53BP1 antibodies (red). Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Survival rate of HeLa cells from (C) after control or CPT treatment was assessed by colony survival assay. Error bars indicate SEM (n = 3).
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Figure 5: Microfluidic platform for cell phenotype and gene function analysis.(A) MCF7 cells delivered with plasmids encoding only Cas9 protein or both sgPten and Cas9 protein were cultured for 7 days and then analyzed by Western blotting with the indicated antibodies. Actin was used as a loading control. The symbol * indicates long exposure. (B) Cells (5 × 104) from (A) were seeded in 60-mm dishes in complete medium and cultured for 7 days. Cells were trypsinized and collected for cell count in a Countess II FL Automated Cell Counter (Life Technologies) daily for 7 days. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (C) HeLa cells delivered with plasmids encoding only Cas9 protein or both sg53BP1 and Cas9 protein were cultured 7 days. Then, the cells were treated with 1 μM CPT for 2 hours and then examined by immunostaining with anti-53BP1 antibodies (red). Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Survival rate of HeLa cells from (C) after control or CPT treatment was assessed by colony survival assay. Error bars indicate SEM (n = 3).

Mentions: Next, we explored gene function and cell phenotype via our delivery chip. Plasmids encoding Cas9 and sgRNA targeting phosphatase and tensin homolog (Pten) (fig. S6A) were delivered into MCF7 cells, followed by culture for 48 hours and puromycin selection. More than 80% of the cells survived the selection process, indicating the high delivery efficiency of our method. Cells were allowed to recover for 7 days and then analyzed by Western blotting. The results of Western blotting analysis showed that endogenous Pten expression was abolished compared with expression in control cells transfected only with plasmid encoding Cas9. Moreover, the level of Akt phosphorylation increased with Pten depletion, consistent with activation of Akt by loss of Pten (Fig. 5A). Cells were immunostained to further confirm successful knockout of Pten and Akt activation (fig. S6B). Cell proliferation was also increased in MCF7 cells after Pten knockout (Fig. 5B), which is consistent with a previous study (20).


CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation.

Han X, Liu Z, Jo MC, Zhang K, Li Y, Zeng Z, Li N, Zu Y, Qin L - Sci Adv (2015)

Microfluidic platform for cell phenotype and gene function analysis.(A) MCF7 cells delivered with plasmids encoding only Cas9 protein or both sgPten and Cas9 protein were cultured for 7 days and then analyzed by Western blotting with the indicated antibodies. Actin was used as a loading control. The symbol * indicates long exposure. (B) Cells (5 × 104) from (A) were seeded in 60-mm dishes in complete medium and cultured for 7 days. Cells were trypsinized and collected for cell count in a Countess II FL Automated Cell Counter (Life Technologies) daily for 7 days. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (C) HeLa cells delivered with plasmids encoding only Cas9 protein or both sg53BP1 and Cas9 protein were cultured 7 days. Then, the cells were treated with 1 μM CPT for 2 hours and then examined by immunostaining with anti-53BP1 antibodies (red). Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Survival rate of HeLa cells from (C) after control or CPT treatment was assessed by colony survival assay. Error bars indicate SEM (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4643799&req=5

Figure 5: Microfluidic platform for cell phenotype and gene function analysis.(A) MCF7 cells delivered with plasmids encoding only Cas9 protein or both sgPten and Cas9 protein were cultured for 7 days and then analyzed by Western blotting with the indicated antibodies. Actin was used as a loading control. The symbol * indicates long exposure. (B) Cells (5 × 104) from (A) were seeded in 60-mm dishes in complete medium and cultured for 7 days. Cells were trypsinized and collected for cell count in a Countess II FL Automated Cell Counter (Life Technologies) daily for 7 days. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (C) HeLa cells delivered with plasmids encoding only Cas9 protein or both sg53BP1 and Cas9 protein were cultured 7 days. Then, the cells were treated with 1 μM CPT for 2 hours and then examined by immunostaining with anti-53BP1 antibodies (red). Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Survival rate of HeLa cells from (C) after control or CPT treatment was assessed by colony survival assay. Error bars indicate SEM (n = 3).
Mentions: Next, we explored gene function and cell phenotype via our delivery chip. Plasmids encoding Cas9 and sgRNA targeting phosphatase and tensin homolog (Pten) (fig. S6A) were delivered into MCF7 cells, followed by culture for 48 hours and puromycin selection. More than 80% of the cells survived the selection process, indicating the high delivery efficiency of our method. Cells were allowed to recover for 7 days and then analyzed by Western blotting. The results of Western blotting analysis showed that endogenous Pten expression was abolished compared with expression in control cells transfected only with plasmid encoding Cas9. Moreover, the level of Akt phosphorylation increased with Pten depletion, consistent with activation of Akt by loss of Pten (Fig. 5A). Cells were immunostained to further confirm successful knockout of Pten and Akt activation (fig. S6B). Cell proliferation was also increased in MCF7 cells after Pten knockout (Fig. 5B), which is consistent with a previous study (20).

Bottom Line: It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9.This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium.We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA. ; Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065, USA.

ABSTRACT
The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9-mediated knockin system.

No MeSH data available.


Related in: MedlinePlus