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Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA - Nucleic Acids Res. (2014)

Bottom Line: To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method.Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells.This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.

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Delivery of a multiplex CRISPR/Cas9 system with a single lentivirus. (A) Four sgRNAs targeting distinct genomic loci were cloned into a lentiviral vector expressing the active Cas9 nuclease. (B) HEK293T cells and primary human dermal fibroblasts were transduced with lentivirus expressing the indicated sgRNAs and assayed for nuclease activity using the Surveyor assay. HEK293T cells were assayed 7 days post-transduction. The human fibroblasts were assayed 10 days post-transduction. (C) Summary of gene editing events at four targeted loci in 10 single cell-derived clonal populations of HEK293T cells transduced with a single lentiviral Cas9 vector. Surveyor analysis for each individual clone is shown in Supplementary Figure S8.
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Figure 3: Delivery of a multiplex CRISPR/Cas9 system with a single lentivirus. (A) Four sgRNAs targeting distinct genomic loci were cloned into a lentiviral vector expressing the active Cas9 nuclease. (B) HEK293T cells and primary human dermal fibroblasts were transduced with lentivirus expressing the indicated sgRNAs and assayed for nuclease activity using the Surveyor assay. HEK293T cells were assayed 7 days post-transduction. The human fibroblasts were assayed 10 days post-transduction. (C) Summary of gene editing events at four targeted loci in 10 single cell-derived clonal populations of HEK293T cells transduced with a single lentiviral Cas9 vector. Surveyor analysis for each individual clone is shown in Supplementary Figure S8.

Mentions: To validate the independent activity of each sgRNA, we assembled a single lentiviral vector expressing active Cas9 and four sgRNAs, each targeting independent loci (Figure 3A). As control vectors, we assembled constructs expressing only one sgRNA along with empty sgRNA expression cassettes in the other three positions. We transduced HEK293Ts and primary fibroblasts with lentiviral vectors expressing the indicated sgRNAs and monitored gene modification frequencies at 7 or 10 days post-transduction, respectively (Figure 3B). In both cell types, the single lentiviral vector mediated highly efficient multiplex editing at all four loci (Figure 3B). Interestingly, expression of all four sgRNAs together resulted in higher modification frequencies than a single sgRNA alone at three out of four loci in fibroblasts (Figure 3B). We observed efficient multiplex gene editing in transduced fibroblasts, which are typically difficult to transfect. Notably, lentiviral transduction of HEK293Ts rapidly introduces gene modifications that are stable for several weeks in culture even in the presence of constitutive Cas9 nuclease expression (Supplementary Figures S5 and S6A–B). A potential concern with this lentiviral system is that the multiple copies of the sgRNA cassettes may spontaneously recombine due to the repetitive sequences in the 3′ end of the sgRNA. To determine the stability of the sgRNA expression cassettes in the integrated viral vector, PCR of the genomic DNA was performed at each time point. PCR bands consistent with full-length sgRNA cassette transfer were detected in the bulk population of transduced HEK293T cells and primary human fibroblasts for up to three weeks (Supplementary Figures S1, S6C and S7). In order to conclusively demonstrate that multiplex gene editing was occurring in single cells, clonal populations were derived from the polyclonal cell population transduced with the construct carrying four sgRNAs (Figure 3A). Ten clonal populations were assessed by the Surveyor assay for gene modification, of which three showed the expected gene editing events at all four loci (Figure 3C and Supplementary Figure S8). Notably, eight of the clones showed editing at two or more loci, indicating that the bulk Surveyor results (Supplementary Figure S6A–B) may be underrepresenting overall editing frequencies after prolonged culture. These data demonstrate that a single lentivirus can express four active sgRNAs efficiently and that this lentiviral platform can be used to target four distinct loci for multiplex CRISPR/Cas9 gene editing.


Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA - Nucleic Acids Res. (2014)

Delivery of a multiplex CRISPR/Cas9 system with a single lentivirus. (A) Four sgRNAs targeting distinct genomic loci were cloned into a lentiviral vector expressing the active Cas9 nuclease. (B) HEK293T cells and primary human dermal fibroblasts were transduced with lentivirus expressing the indicated sgRNAs and assayed for nuclease activity using the Surveyor assay. HEK293T cells were assayed 7 days post-transduction. The human fibroblasts were assayed 10 days post-transduction. (C) Summary of gene editing events at four targeted loci in 10 single cell-derived clonal populations of HEK293T cells transduced with a single lentiviral Cas9 vector. Surveyor analysis for each individual clone is shown in Supplementary Figure S8.
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Figure 3: Delivery of a multiplex CRISPR/Cas9 system with a single lentivirus. (A) Four sgRNAs targeting distinct genomic loci were cloned into a lentiviral vector expressing the active Cas9 nuclease. (B) HEK293T cells and primary human dermal fibroblasts were transduced with lentivirus expressing the indicated sgRNAs and assayed for nuclease activity using the Surveyor assay. HEK293T cells were assayed 7 days post-transduction. The human fibroblasts were assayed 10 days post-transduction. (C) Summary of gene editing events at four targeted loci in 10 single cell-derived clonal populations of HEK293T cells transduced with a single lentiviral Cas9 vector. Surveyor analysis for each individual clone is shown in Supplementary Figure S8.
Mentions: To validate the independent activity of each sgRNA, we assembled a single lentiviral vector expressing active Cas9 and four sgRNAs, each targeting independent loci (Figure 3A). As control vectors, we assembled constructs expressing only one sgRNA along with empty sgRNA expression cassettes in the other three positions. We transduced HEK293Ts and primary fibroblasts with lentiviral vectors expressing the indicated sgRNAs and monitored gene modification frequencies at 7 or 10 days post-transduction, respectively (Figure 3B). In both cell types, the single lentiviral vector mediated highly efficient multiplex editing at all four loci (Figure 3B). Interestingly, expression of all four sgRNAs together resulted in higher modification frequencies than a single sgRNA alone at three out of four loci in fibroblasts (Figure 3B). We observed efficient multiplex gene editing in transduced fibroblasts, which are typically difficult to transfect. Notably, lentiviral transduction of HEK293Ts rapidly introduces gene modifications that are stable for several weeks in culture even in the presence of constitutive Cas9 nuclease expression (Supplementary Figures S5 and S6A–B). A potential concern with this lentiviral system is that the multiple copies of the sgRNA cassettes may spontaneously recombine due to the repetitive sequences in the 3′ end of the sgRNA. To determine the stability of the sgRNA expression cassettes in the integrated viral vector, PCR of the genomic DNA was performed at each time point. PCR bands consistent with full-length sgRNA cassette transfer were detected in the bulk population of transduced HEK293T cells and primary human fibroblasts for up to three weeks (Supplementary Figures S1, S6C and S7). In order to conclusively demonstrate that multiplex gene editing was occurring in single cells, clonal populations were derived from the polyclonal cell population transduced with the construct carrying four sgRNAs (Figure 3A). Ten clonal populations were assessed by the Surveyor assay for gene modification, of which three showed the expected gene editing events at all four loci (Figure 3C and Supplementary Figure S8). Notably, eight of the clones showed editing at two or more loci, indicating that the bulk Surveyor results (Supplementary Figure S6A–B) may be underrepresenting overall editing frequencies after prolonged culture. These data demonstrate that a single lentivirus can express four active sgRNAs efficiently and that this lentiviral platform can be used to target four distinct loci for multiplex CRISPR/Cas9 gene editing.

Bottom Line: To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method.Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells.This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.

Show MeSH