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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

Gene disruption via chip.Plasmids encoding both sgRNA targeting AAVS1 locus or NUAK2 and Cas9 protein were delivered into MCF7 and HeLa cells, respectively. After 7 days of cell culture, genomic DNA was extracted. PCR product sequencing for specific targeting regions was performed. (A) PCR product sequencing data for the sgAAVS1 targeting region. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. Representative sequences for indels are shown. Short black lines denote different deletions. Black arrow denotes an insertion. (B) Surveyor mutation detection assay for sgAAVS1- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S. The symbol * indicates the cleavage lane of DNA bands after cells went through the same chip three times. (C) Illustration of sgNUAK2 targeting region at the first exon. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. (D) PCR product sequencing data for the sgNUAK2 targeting region. Representative sequences for deletions are shown. Short black lines denote different deletions. (E) Surveyor mutation detection assay for sgNUAK2- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S.
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Figure 4: Gene disruption via chip.Plasmids encoding both sgRNA targeting AAVS1 locus or NUAK2 and Cas9 protein were delivered into MCF7 and HeLa cells, respectively. After 7 days of cell culture, genomic DNA was extracted. PCR product sequencing for specific targeting regions was performed. (A) PCR product sequencing data for the sgAAVS1 targeting region. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. Representative sequences for indels are shown. Short black lines denote different deletions. Black arrow denotes an insertion. (B) Surveyor mutation detection assay for sgAAVS1- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S. The symbol * indicates the cleavage lane of DNA bands after cells went through the same chip three times. (C) Illustration of sgNUAK2 targeting region at the first exon. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. (D) PCR product sequencing data for the sgNUAK2 targeting region. Representative sequences for deletions are shown. Short black lines denote different deletions. (E) Surveyor mutation detection assay for sgNUAK2- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S.

Mentions: To determine whether our delivery platform could be used for gene disruption and function analysis, we carried out further delivery of plasmids encoding Cas9 and sgRNAs targeting different genes in different types of cell lines. Plasmids encoding sgRNA targeting the endogenous AAVS1 locus and Cas9 were delivered into MCF7 cells. The cells were allowed to recover in culture for 7 days, followed by PCR amplification of the specific sgRNA target region. The results of TA cloning and sequence analysis showed that the delivery of plasmids encoding Cas9 and sgRNA targeting AAVS1 resulted in mutations, including indels, at the specific genomic loci (Fig. 4A). Surveyor mutation detection assay revealed substantial cleavage at the AAVS1 locus, with indels occurring at a frequency of about 18 to 46% when delivery was optimized by passage of the cells through the chip three times (Fig. 4B).


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)

Gene disruption via chip.Plasmids encoding both sgRNA targeting AAVS1 locus or NUAK2 and Cas9 protein were delivered into MCF7 and HeLa cells, respectively. After 7 days of cell culture, genomic DNA was extracted. PCR product sequencing for specific targeting regions was performed. (A) PCR product sequencing data for the sgAAVS1 targeting region. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. Representative sequences for indels are shown. Short black lines denote different deletions. Black arrow denotes an insertion. (B) Surveyor mutation detection assay for sgAAVS1- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S. The symbol * indicates the cleavage lane of DNA bands after cells went through the same chip three times. (C) Illustration of sgNUAK2 targeting region at the first exon. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. (D) PCR product sequencing data for the sgNUAK2 targeting region. Representative sequences for deletions are shown. Short black lines denote different deletions. (E) Surveyor mutation detection assay for sgNUAK2- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Gene disruption via chip.Plasmids encoding both sgRNA targeting AAVS1 locus or NUAK2 and Cas9 protein were delivered into MCF7 and HeLa cells, respectively. After 7 days of cell culture, genomic DNA was extracted. PCR product sequencing for specific targeting regions was performed. (A) PCR product sequencing data for the sgAAVS1 targeting region. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. Representative sequences for indels are shown. Short black lines denote different deletions. Black arrow denotes an insertion. (B) Surveyor mutation detection assay for sgAAVS1- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S. The symbol * indicates the cleavage lane of DNA bands after cells went through the same chip three times. (C) Illustration of sgNUAK2 targeting region at the first exon. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. (D) PCR product sequencing data for the sgNUAK2 targeting region. Representative sequences for deletions are shown. Short black lines denote different deletions. (E) Surveyor mutation detection assay for sgNUAK2- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S.
Mentions: To determine whether our delivery platform could be used for gene disruption and function analysis, we carried out further delivery of plasmids encoding Cas9 and sgRNAs targeting different genes in different types of cell lines. Plasmids encoding sgRNA targeting the endogenous AAVS1 locus and Cas9 were delivered into MCF7 cells. The cells were allowed to recover in culture for 7 days, followed by PCR amplification of the specific sgRNA target region. The results of TA cloning and sequence analysis showed that the delivery of plasmids encoding Cas9 and sgRNA targeting AAVS1 resulted in mutations, including indels, at the specific genomic loci (Fig. 4A). Surveyor mutation detection assay revealed substantial cleavage at the AAVS1 locus, with indels occurring at a frequency of about 18 to 46% when delivery was optimized by passage of the cells through the chip three times (Fig. 4B).

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