Limits...
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.

Show MeSH
Stable gene activation by dCas9VP64 in HEK293T cells using a single multiplex lentiviral vector. HEK293T cells were transduced with lentivirus to stably express dCas9VP64 and the indicated combinations of sgRNAs. Activation of the endogenous (A) IL1RN and (B) HBG1 loci at 7 days post-transduction were tunable by varying the number of sgRNAs delivered (*P < 0.05 versus no sgRNAs). Peak levels of endogenous (C) IL1RN and (D) HBG1 were observed 6 days post-transduction and the level of activation was sustained through day 21.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231726&req=5

Figure 5: Stable gene activation by dCas9VP64 in HEK293T cells using a single multiplex lentiviral vector. HEK293T cells were transduced with lentivirus to stably express dCas9VP64 and the indicated combinations of sgRNAs. Activation of the endogenous (A) IL1RN and (B) HBG1 loci at 7 days post-transduction were tunable by varying the number of sgRNAs delivered (*P < 0.05 versus no sgRNAs). Peak levels of endogenous (C) IL1RN and (D) HBG1 were observed 6 days post-transduction and the level of activation was sustained through day 21.

Mentions: Lentiviral delivery may enable stable, long-term gene activation by CRISPR/Cas9 transactivation. To test this, HEK293Ts were transduced using a single lentiviral vector encoding dCas9VP64 and one to four sgRNA expression cassettes. Similar to the transient transfection results (Figure 4), we were able to tunably and robustly activate expression of endogenous IL1RN and HBG1 genes (Figure 5A and B). Gene activation induced by co-transfection of HEK293T cells with dCas9VP64 and four sgRNAs targeted to the IL1RN and HBG1 promoters peaked between three and five days post-transfection and gene expression returned to background levels 15–20 days post-transfection (Figure 4C and D). In contrast, lentiviral delivery of dCas9VP64 and the same four IL1RN or HBG1-targeted sgRNAs induced sustained gene activation for more than 20 days post-transduction (Figure 5C and D). Thus, single lentiviral delivery of multiplex dCas9VP64 transactivators is a useful platform to efficiently and stably upregulate endogenous target genes.


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 HEK293T cells using a single multiplex lentiviral vector. HEK293T cells were transduced with lentivirus to stably express dCas9VP64 and the indicated combinations of sgRNAs. Activation of the endogenous (A) IL1RN and (B) HBG1 loci at 7 days post-transduction were tunable by varying the number of sgRNAs delivered (*P < 0.05 versus no sgRNAs). Peak levels of endogenous (C) IL1RN and (D) HBG1 were observed 6 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 5: Stable gene activation by dCas9VP64 in HEK293T cells using a single multiplex lentiviral vector. HEK293T cells were transduced with lentivirus to stably express dCas9VP64 and the indicated combinations of sgRNAs. Activation of the endogenous (A) IL1RN and (B) HBG1 loci at 7 days post-transduction were tunable by varying the number of sgRNAs delivered (*P < 0.05 versus no sgRNAs). Peak levels of endogenous (C) IL1RN and (D) HBG1 were observed 6 days post-transduction and the level of activation was sustained through day 21.
Mentions: Lentiviral delivery may enable stable, long-term gene activation by CRISPR/Cas9 transactivation. To test this, HEK293Ts were transduced using a single lentiviral vector encoding dCas9VP64 and one to four sgRNA expression cassettes. Similar to the transient transfection results (Figure 4), we were able to tunably and robustly activate expression of endogenous IL1RN and HBG1 genes (Figure 5A and B). Gene activation induced by co-transfection of HEK293T cells with dCas9VP64 and four sgRNAs targeted to the IL1RN and HBG1 promoters peaked between three and five days post-transfection and gene expression returned to background levels 15–20 days post-transfection (Figure 4C and D). In contrast, lentiviral delivery of dCas9VP64 and the same four IL1RN or HBG1-targeted sgRNAs induced sustained gene activation for more than 20 days post-transduction (Figure 5C and D). Thus, single lentiviral delivery of multiplex dCas9VP64 transactivators is a useful platform to efficiently and stably upregulate endogenous target genes.

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