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. 2017 Dec;3(12):930-936.
doi: 10.1038/s41477-017-0046-0. Epub 2017 Nov 20.

A Potent Cas9-derived Gene Activator for Plant and Mammalian Cells

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Free PMC article

A Potent Cas9-derived Gene Activator for Plant and Mammalian Cells

Zhenxiang Li et al. Nat Plants. .
Free PMC article

Abstract

Overexpression of complementary DNA represents the most commonly used gain-of-function approach for interrogating gene functions and for manipulating biological traits. However, this approach is challenging and inefficient for multigene expression due to increased labour for cloning, limited vector capacity, requirement of multiple promoters and terminators, and variable transgene expression levels. Synthetic transcriptional activators provide a promising alternative strategy for gene activation by tethering an autonomous transcription activation domain (TAD) to an intended gene promoter at the endogenous genomic locus through a programmable DNA-binding module. Among the known custom DNA-binding modules, the nuclease-dead Streptococcus pyogenes Cas9 (dCas9) protein, which recognizes a specific DNA target through base pairing between a synthetic guide RNA and DNA, outperforms zinc-finger proteins and transcription activator-like effectors, both of which target through protein-DNA interactions 1 . Recently, three potent dCas9-based transcriptional activation systems, namely VPR, SAM and SunTag, have been developed for animal cells 2-6 . However, an efficient dCas9-based transcriptional activation platform is still lacking for plant cells 7-9 . Here, we developed a new potent dCas9-TAD, named dCas9-TV, through plant cell-based screens. dCas9-TV confers far stronger transcriptional activation of single or multiple target genes than the routinely used dCas9-VP64 activator in both plant and mammalian cells.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors have filed a patent application based on some results reported in this paper.

Figures

Figure 1
Figure 1
dCas9-TV-mediated gene activation in Arabidopsis. (a) Diagram of three representative dCas9 gene activators tested in this study. (b) dCas9-VP128 exhibits the highest activity among dCas9-(VP64)n (n=1-4) activators in Arabidopsis protoplasts. dCas9-VP256 is marked by an asterisk in the blot. (c) dCas9-6TAL-VP128 (dCas9-TV) exhibits the highest activity among all screened dCas9 activators in Arabidopsis protoplasts. dCas9-8TAL-VP128 is marked by an asterisk in the blot. (d) Representative transgenic Arabidopsis T1 plants co-expressing dCas9-TV and sgRNA-WRKY30 show strong induction of endogenous WRKY30. (e, f) Arabidopsis protoplasts (e) or representative transgenic T2 plants (f) co-expressing dCas9-TV and sgRNA-RLP23 show robust induction of endogenous RLP23. (g, h) Arabidopsis transgenic T2 plants with RLP23 induction accordingly exhibit enhanced MAPK activation (g) and ROS production (h) in response to the pathogen elicitor nlp20. RuBisCo in (g) indicates equal protein loading. (i) Evaluation of dCas9-TV specificity by RNA-seq. Arabidopsis protoplasts expressing or not expressing dCas9-TV and sgRNA-RLP23 exhibit very similar transcriptome profiles. R indicates Pearson’s correlation coefficient. RLP23 is marked by a red dot close to the y-axis as a highly activated gene. (j) Co-expression of dCas9-TV and 3 sgRNAs leads to simultaneous induction of WRKY30, RLP23 and CDG1 in Arabidopsis protoplasts. Empty vectors were used in Control (Ctrl) samples to replace constructs expressing dCas9 activator and sgRNAs. Gene activation was determined by promoter-LUC assays (b, c) or RT-qPCR (d-f, j), and data are shown as mean (indicated with a number on the top) ± sd (n=3). *P < 0.05, **P < 0.01 (student’s t-test). n.s., not significant.
Figure 2
Figure 2
Modified strategies of dCas9-TV-mediated gene activation in Arabidopsis and other cells. (a) Diagram of the SAM sgRNA, MCP activators and their interactions. (b) SAM sgRNA-associated MCP activators fail to strengthen dCas9-TV-mediated WRKY30 activation in Arabidopsis protoplasts. (c, d) Rice protoplasts co-expressing dCas9-TV and sgRNAs targeting to GW7 (c) or ER1 (d) exhibit strong target gene induction. (e) Co-expression of dCas9-TV and 2 sgRNAs leads to simultaneous induction of GW7 and ER1 in rice protoplasts. (f) HEK 293T cells co-expressing dCas9-TV and sgRNAs targeting to ASCL1 or OCT4 exhibit clear induction of these genes. (g) dCas9-TV ribonucleoprotein (RNP)-mediated activation of WRKY30 and RLP23 in Arabidopsis protoplasts and ER1 in rice protoplasts. Empty vectors were used in Control (Ctrl) samples to replace constructs expressing dCas9 activator and sgRNAs. Gene activation was determined by promoter-LUC assays (b-d) or RT-qPCR (e-g), and data are shown as mean (indicated by a number on the top) ± sd (n=3). n.s., not significant.

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