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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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Stable gene activation by dCas9VP64 in primary human fibroblasts using a single multiplex lentiviral vector. Primary human dermal fibroblasts were transduced with lentivirus to stably co-express dCas9VP64 and four sgRNAs targeted to the IL1RN promoter. Peak levels of endogenous IL1RN were observed 14 days post-transduction and the level of activation was sustained through day 21.
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Figure 6: Stable gene activation by dCas9VP64 in primary human fibroblasts using a single multiplex lentiviral vector. Primary human dermal fibroblasts were transduced with lentivirus to stably co-express dCas9VP64 and four sgRNAs targeted to the IL1RN promoter. Peak levels of endogenous IL1RN were observed 14 days post-transduction and the level of activation was sustained through day 21.

Mentions: Many applications require long-term stable gene expression in primary cells that are difficult to transfect. To test the lentiviral vector in this context, we transduced primary human dermal fibroblasts with a single lentivirus co-expressing dCas9VP64 and four sgRNAs targeted against the IL1RN or HBG1 endogenous promoters. We observed sustained activation of the IL1RN locus for up to 21 days post-transduction (Figure 6). However, we did not observe any activation of the HBG1 locus (data not shown). This may be because the HBG1 sgRNAs induce lower levels of gene activation compared to the IL1RN sgRNAs (Figure 5C and D). Furthermore, activation in the primary dermal fibroblasts was notably lower than in HEK293T cells (Figures 5C and 6), potentially due to lower overall expression levels or epigenetic context. While our platform provides a method for sustained expression of the CRISPR/Cas9 components, these results highlight the importance of identifying potent combinations of sgRNAs to induce activation in diverse cell types.


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

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

Stable gene activation by dCas9VP64 in primary human fibroblasts using a single multiplex lentiviral vector. Primary human dermal fibroblasts were transduced with lentivirus to stably co-express dCas9VP64 and four sgRNAs targeted to the IL1RN promoter. Peak levels of endogenous IL1RN were observed 14 days post-transduction and the level of activation was sustained through day 21.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231726&req=5

Figure 6: Stable gene activation by dCas9VP64 in primary human fibroblasts using a single multiplex lentiviral vector. Primary human dermal fibroblasts were transduced with lentivirus to stably co-express dCas9VP64 and four sgRNAs targeted to the IL1RN promoter. Peak levels of endogenous IL1RN were observed 14 days post-transduction and the level of activation was sustained through day 21.
Mentions: Many applications require long-term stable gene expression in primary cells that are difficult to transfect. To test the lentiviral vector in this context, we transduced primary human dermal fibroblasts with a single lentivirus co-expressing dCas9VP64 and four sgRNAs targeted against the IL1RN or HBG1 endogenous promoters. We observed sustained activation of the IL1RN locus for up to 21 days post-transduction (Figure 6). However, we did not observe any activation of the HBG1 locus (data not shown). This may be because the HBG1 sgRNAs induce lower levels of gene activation compared to the IL1RN sgRNAs (Figure 5C and D). Furthermore, activation in the primary dermal fibroblasts was notably lower than in HEK293T cells (Figures 5C and 6), potentially due to lower overall expression levels or epigenetic context. While our platform provides a method for sustained expression of the CRISPR/Cas9 components, these results highlight the importance of identifying potent combinations of sgRNAs to induce activation in diverse cell types.

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