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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

Governing parameters and broad applicability.(A) Microscopy of HEK293T cells into which FITC-labeled ssDNA was delivered through our chip. Results shown are from two independent chips. Control indicated all the same treatments for the cells except passing through the chip. Scale bar, 50 μm. BF, bright field. (B and C) Delivery efficiency (B) and cell viability (C) 16 hours after treatment were calculated for (A) as a function of fluid speed at different parameter designs; 4 or 5 μm indicates the constriction width, and 4 μm ×3 indicates cells passing through the same device three times. Error bars indicate SEM (n = 3). (D) Western blotting of PC-3 cells 48 hours after delivery with three different siRNA oligos targeting Akt1. Actin is showed as a loading control. (E) Cells from (D) were seeded in complete medium and, after 6 days, were recovered and trypsinized to count the numbers with a Countess II FL Automated Cell Counter (Life Technologies). Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (F) Delivery efficiency in different cell lines. HEK293T cells, human luminal-like MCF7 and basal-like SUM159 breast cancer cells, human SU-DHL-1 anaplastic large cell lymphoma cells, and mouse AB2.2 embryonic stem cells were delivered with plasmids encoding GFP. Untreated serves as a negative control and FuGENE HD serves as a positive control. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test.
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Figure 2: Governing parameters and broad applicability.(A) Microscopy of HEK293T cells into which FITC-labeled ssDNA was delivered through our chip. Results shown are from two independent chips. Control indicated all the same treatments for the cells except passing through the chip. Scale bar, 50 μm. BF, bright field. (B and C) Delivery efficiency (B) and cell viability (C) 16 hours after treatment were calculated for (A) as a function of fluid speed at different parameter designs; 4 or 5 μm indicates the constriction width, and 4 μm ×3 indicates cells passing through the same device three times. Error bars indicate SEM (n = 3). (D) Western blotting of PC-3 cells 48 hours after delivery with three different siRNA oligos targeting Akt1. Actin is showed as a loading control. (E) Cells from (D) were seeded in complete medium and, after 6 days, were recovered and trypsinized to count the numbers with a Countess II FL Automated Cell Counter (Life Technologies). Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (F) Delivery efficiency in different cell lines. HEK293T cells, human luminal-like MCF7 and basal-like SUM159 breast cancer cells, human SU-DHL-1 anaplastic large cell lymphoma cells, and mouse AB2.2 embryonic stem cells were delivered with plasmids encoding GFP. Untreated serves as a negative control and FuGENE HD serves as a positive control. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test.

Mentions: To optimize the delivery performance of the chip, we took into consideration constriction dimensions, fluid flow rates, and duration of cell passage through the chip as three key parameters. In the diamond design, the constriction depth was 15 μm, and the width varied from 4 to 5 μm (Fig. 1C). In pursuit of high delivery efficiency coupled with high cell viability, we did a series of testing deliveries of FITC-labeled ssDNA into HEK293T cells (Fig. 2A). Our data showed that delivery efficiency increased with increasing flow rate across design patterns (Fig. 2B). The 4-μm constriction width presented higher delivery efficiency than the 5-μm width at all flow rates, with minimal effect on cell viability. Increasing the number of operational cycles with the same chip allowed multiple cell passaging times, which would also enhance the delivery efficiency; however, the operation clearly decreased cell viability (Fig. 2, B and C). The data for the 0 μl/min flow rate represents a control whereby the cells were treated exactly as the other samples but were not applied with the membrane deformation, thus ruling out the possibility that cell FITC positivity was the result of any endocytotic or surface binding events.


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)

Governing parameters and broad applicability.(A) Microscopy of HEK293T cells into which FITC-labeled ssDNA was delivered through our chip. Results shown are from two independent chips. Control indicated all the same treatments for the cells except passing through the chip. Scale bar, 50 μm. BF, bright field. (B and C) Delivery efficiency (B) and cell viability (C) 16 hours after treatment were calculated for (A) as a function of fluid speed at different parameter designs; 4 or 5 μm indicates the constriction width, and 4 μm ×3 indicates cells passing through the same device three times. Error bars indicate SEM (n = 3). (D) Western blotting of PC-3 cells 48 hours after delivery with three different siRNA oligos targeting Akt1. Actin is showed as a loading control. (E) Cells from (D) were seeded in complete medium and, after 6 days, were recovered and trypsinized to count the numbers with a Countess II FL Automated Cell Counter (Life Technologies). Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (F) Delivery efficiency in different cell lines. HEK293T cells, human luminal-like MCF7 and basal-like SUM159 breast cancer cells, human SU-DHL-1 anaplastic large cell lymphoma cells, and mouse AB2.2 embryonic stem cells were delivered with plasmids encoding GFP. Untreated serves as a negative control and FuGENE HD serves as a positive control. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Governing parameters and broad applicability.(A) Microscopy of HEK293T cells into which FITC-labeled ssDNA was delivered through our chip. Results shown are from two independent chips. Control indicated all the same treatments for the cells except passing through the chip. Scale bar, 50 μm. BF, bright field. (B and C) Delivery efficiency (B) and cell viability (C) 16 hours after treatment were calculated for (A) as a function of fluid speed at different parameter designs; 4 or 5 μm indicates the constriction width, and 4 μm ×3 indicates cells passing through the same device three times. Error bars indicate SEM (n = 3). (D) Western blotting of PC-3 cells 48 hours after delivery with three different siRNA oligos targeting Akt1. Actin is showed as a loading control. (E) Cells from (D) were seeded in complete medium and, after 6 days, were recovered and trypsinized to count the numbers with a Countess II FL Automated Cell Counter (Life Technologies). Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (F) Delivery efficiency in different cell lines. HEK293T cells, human luminal-like MCF7 and basal-like SUM159 breast cancer cells, human SU-DHL-1 anaplastic large cell lymphoma cells, and mouse AB2.2 embryonic stem cells were delivered with plasmids encoding GFP. Untreated serves as a negative control and FuGENE HD serves as a positive control. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test.
Mentions: To optimize the delivery performance of the chip, we took into consideration constriction dimensions, fluid flow rates, and duration of cell passage through the chip as three key parameters. In the diamond design, the constriction depth was 15 μm, and the width varied from 4 to 5 μm (Fig. 1C). In pursuit of high delivery efficiency coupled with high cell viability, we did a series of testing deliveries of FITC-labeled ssDNA into HEK293T cells (Fig. 2A). Our data showed that delivery efficiency increased with increasing flow rate across design patterns (Fig. 2B). The 4-μm constriction width presented higher delivery efficiency than the 5-μm width at all flow rates, with minimal effect on cell viability. Increasing the number of operational cycles with the same chip allowed multiple cell passaging times, which would also enhance the delivery efficiency; however, the operation clearly decreased cell viability (Fig. 2, B and C). The data for the 0 μl/min flow rate represents a control whereby the cells were treated exactly as the other samples but were not applied with the membrane deformation, thus ruling out the possibility that cell FITC positivity was the result of any endocytotic or surface binding events.

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