Positional effects on efficiency of CRISPR/Cas9-based transcriptional activation in rice plants
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The nuclease-dead Cas9 (dCas9) has been reprogrammed for transcriptional activation by fusing dCas9 to a transcriptional activation domain. In the presence of a guide RNA (gRNA), the dCas9 fusions specifically bind to regions of a promoter to activate transcription. Significant amount of effort has been directed toward the identification and optimization of the fusions of dCas9-activation domain, but very little is known about the impact of gRNA target positions within a promoter in plants on transcriptional activation efficiency. The dCas9–6TAL–VP128 system (dCas9-TV) has been optimized to activate transcription in plants. Here we use the dCas9-TV to activate transcription of OsWOX11 and OsYUC1, two genes that cause dramatic developmental phenotypes when overexpressed. We designed a series of gRNAs targeting the promoters of the two genes. We show that gRNAs that target regions within 350 bp upstream of the transcription start site were most effective in transcriptional activation. Moreover, we show that using two gRNAs that simultaneously target two discrete sites in a promoter can further enhance transcription. This work provides guidelines for designed transcriptional activation through CRISPR/dCas9 systems.
Designed transcriptional activation of genes not only provides an effective tool for defining gene functions, but also is a foundation for commercial production of agriculturally and medically valuable products. In plants, genes can be activated when the Cauliflower mosaic virus 35S enhancers (CaMV 35S) are inserted near a gene, which is the basis of the widely used activation-tagging screens (Weigel et al. 2000). However, because gene targeting in plants is still very inefficient, currently it is very difficult to precisely place transcriptional enhancers near a target gene using homology-directed recombination (HDR)-based gene targeting technologies (Li et al. 2019a). A gene can also be overexpressed using transgenic approaches by placing the genomic DNA or cDNA under the control of a strong promoter such as UBIQUITIN, ACTIN, or CaMV35S promoters. But the overexpression constructs are often randomly inserted into a genome, rendering it necessary to characterize multiple transgenic lines to rule out positional effects. There are several inherent obstacles in using transgenic approaches. Multiplexing overexpression of genes is difficult because placing several overexpression cassettes in the same plasmid is time consuming and may render the plasmid unstable. Moreover, large plasmids may also make plant transformation less efficient (Park et al. 2000). It is difficult to obtain a complete cDNA for some large genes such as BIG in Arabidopsis, which has a coding region of 15 kb (Gil et al. 2001).
Cas9 nuclease can be brought to a specific target DNA locus through a guide RNA molecule (gRNA), which forms a duplex with the DNA target through base pairing. Cas9 then generates a double-stranded break (DSB) in the target sequence, providing opportunities for editing the target sequence using HDR or non-homologous end-joining DNA repair mechanisms (Mali et al. 2013). Recently, the CRISPR/Cas9 gene editing system has been repurposed for genomic activation and repression (Gilbert et al. 2013). When the key catalytic residues in Cas9 are mutated, the nuclease-dead Cas9 (dCas9) still retains the ability to bind DNA in a gRNA-dependent manner, but can no longer generate DSB (Qi et al. 2013). The dCas9 has been repurposed by fusing it with various functional domains for transcriptional activation, repression, visualization of cis-elements, epigenetic modification, and other applications (Adli 2018). When dCas9 is fused to a transcriptional activation domain, the fusion protein can be brought to a specific promoter in a genome through a gRNA. Subsequently, transcription of the gene can be activated, leading to strong expression of a target gene (Li et al. 2017; Lowder et al. 2015; Lowder et al. 2018; Piatek et al. 2015; Vazquez-Vilar et al. 2016). CRISPR/Cas9-mediated transcriptional activation offers several advantages. It is relatively straightforward to activate multiple genes using dCas9 because several gRNA multiplexing systems have been well established (He et al. 2017; Ma et al. 2015; Xie et al. 2015). The dCas9/gRNA activation system probably does not cause co-suppression, a phenomenon that is often encountered when transgenic approaches are used (Napoli et al. 1990).
Single transcriptional activation domain and combinations of various activation domains have been fused to dCas9 and the fusion proteins have been evaluated in animal cells and in plants for their efficiency in gRNA-dependent gene activation (Li et al. 2017; Lowder et al. 2015; Piatek et al. 2015; Vazquez-Vilar et al. 2016). The dCas9-6TAL-VP128 system (dCas9-TV), which consists of the combination of VP128 with up to six copies of the TALE transactivation motif, has emerged as a very potent and programmable transcriptional activator (Li et al. 2017, 2019c). It is known that the locations of gRNA targets within the regulatory sequences of a gene have a profound effect on dCas9-based transcriptional activation. In animal cells, it was reported that the optimal window for gRNA targeting is within 400 bp upstream of the transcription start site (TSS) (Gilbert et al. 2014; Konermann et al. 2015). However, it is not clear whether similar regions are also optimal for plant cells. Another consideration is whether to use several gRNAs targeting the same promoter simultaneously. Several gRNAs simultaneously targeting the same promoter in plants using dCas9-VP64 displayed synergistic effects (Lowder et al. 2015; Lowder et al. 2018; Piatek et al. 2015; Vazquez-Vilar et al. 2016). Here, we used the potent dCas9-TV (Li et al. 2017) to activate the transcription of rice OsWOX11 and OsYUC1, whose overexpression phenotypes in rice have been well documented (Yamamoto et al. 2007; Zhang et al. 2018; Zhao et al. 2009).
We tested whether additional gRNAs can enhance transcriptional activation. We added a second gRNA to the plasmids that can express either OsWOX11 gRNA1 or gRNA3, which displayed the most efficient transcriptional activation (Fig. 1B, D). As shown in Fig. 1, the addition of gRNA8, which targeted a region around − 600 bp (Fig. 1A), led to more than tenfold increase of OsWOX11 expression compared to gRNA1 alone (Fig. 1B, D). The gRNA6 and gRNA10 only led to a slight increase of OsWOX11 expression (Fig. 1B). Without a gRNA that targeted the core region (within 350 bp upstream of the TSS), even two gRNAs were not effective to induce OsWOX11 expression (Fig. 1A, B).
We also targeted the promoter of OsYUC1, which encodes a flavin-containing monooxygenase and which plays a key role in auxin biosynthesis (Yamamoto et al. 2007; Zhao et al. 2001). We designed five gRNAs that target regions in the OsYUC1 promoter (Fig. 1E). Overexpression of OsYUC1 is known to promote adventitious root formation out of coleoptiles (Yamamoto et al. 2007; Zhang et al. 2018) (Fig. 1F). When OsYUC1 was overexpressed, many hairy roots were initiated from the coleoptile (Fig. 1F). It was also clear that overexpression of OsYUC1 inhibited the primary root elongation (Fig. 1F). Introduction of dCas9-TV system and the gRNA1, which targets the TG1 (Fig. 1E), into rice led to development of adventitious roots out of coleoptiles (Fig. 1F). Note that the TG1 plants had two clusters of roots and two apparent shoots (Fig. 1F). The root-like structure that links the two clusters of roots was actually coleoptile, from which adventitious roots were also developed (Fig. 1F). Both TG1 plants and Ubi-OsYUC1 plants developed more adventitious roots. Interestingly, the TG1 plants displayed root phenotypes that were different from Ubi-OsYUC1 plants (Fig. 1F), which was likely caused by differences in timing and magnitude of OsYUC1 overexpression. Consistent with the observed phenotypes, we found that the gRNA1 caused more than 1000-fold increases in OsYUC1 expression (Fig. 1F, G). Similar to our observations with OsWOX11 constructs, the gRNA that targeted the region within 350 bp upstream of the TSS of OsYUC1 was the most effective (Fig. 1E–G). Interestingly, adding another gRNA (gRNA2) did not significantly affect the expression of OsYUC1 (Fig. 1F, G). Again, without a gRNA targeting the core region, even two gRNAs were not effective for activating OsYUC1 expression (Fig. 1E, F).
We have shown clearly that dCas9-TV could effectively activate gene transcription in stable transgenic rice plants. Our work suggests that the most effective target regions are within the 350 bp upstream of the TSS. Traditionally, gRNAs for dCas9-TV-based transcriptional activation are first optimized using plant protoplast systems (Li et al. 2019b). Our findings that regions 350 bp upstream of the TSS are most effective for dCas9-TV-based transcriptional activation indicate that it can be productive by focusing on the small region without using the protoplast systems. Moreover, we demonstrated that simultaneously target two regions within the same promoter may be beneficial. At least, we did not observe strong detrimental effects when two gRNAs are simultaneously employed. Analyzing additional gRNA targets and tiling the entire promoters of OsWOX11 and OsYUC1 will help refine the regions and sequences optimal for dCas9-mediated transcriptional activation.
Materials and methods
Construction of transcriptional activation vectors
The pCAMBIA-dCas9-TV plasmid from Prof. Jian-Feng Li at the Sun Yat-Sen University provides the backbone of all the plasmids used in this study. For gRNA constructs, the gRNA expression cassette was inserted into the SwaI site of the dCas9-TV vector. For plasmids that express two gRNAs, one gRNA was driven by the OsU6 promoter and the other was under the control of the OsU3 promoter. The location and sequences of gRNAs used in this study are listed in the Supplementary Table 2. The CRISPR P 2.0 (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR) was used for selecting gRNA targets (Liu et al. 2017).
Wild-type rice (Oryza sativa ssp. japonica) “Zhonghua 11” (ZH11) was used in this study. ZH11 was transformed via Agrobacterium tumefaciens (EHA105)-mediated callus transformation as previously described (Hiei et al. 1994).
RNA extraction and RT-PCR analysis
Young leaves of plants were collected and total RNA was extracted using TRIzol reagent (Invitrogen). Complementary DNA was synthesized according to the manufacturer’s instructions (Invitrogen). RT-qPCRs were performed using gene-specific primers (Supplementary Table 3) and SYBR Premix Ex-Taq in a real-time PCR 7500 system (Applied Biosystems). Data were collected using the ABI PRISM 7500 sequence detection system following the manufacturer’s instruction. The rice ACTIN1 gene was used as the internal control. At least three technical repeats were conducted.
Sequence data of rice genes in this article are accessible at the TIGR (Rice Genome Annotation Project http://rice.plantbiology.msu.edu) with the following accession numbers: OsYUC1, LOC_Os01g45760; OsWOX11, LOC_Os07g48560.
We thank Professor Jian-Feng Li for providing plasmids. This work was supported by a National Transgenic Science and Technology Program (2016ZX08010002) to R.W.
YZ conceived the study. YZ and TZ designed the experiments. XG, TZ and JX performed the experiments. TZ and YZ wrote the manuscript.
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