Improvement of protoplast transfection conditions
To develop a robust CRISPRa and CRISPRi system for maize protoplasts, we first analyzed the following protoplast transfection conditions. Protoplasts were isolated from two-week-old etiolated maize seedlings as described in Burdo et al. (2014). Next, we compared electroporation with PEG-mediated transfection, where 40% PEG was prepared in either 0.2 M or 0.4 M mannitol as described for Arabidopsis and rice protoplast transfection, respectively [24,30]. After transfection with a construct that carried a GFP-expression cassette (pCXUN-HA-GFP, Additional File 2), we conducted western blot analysis. As shown in Figure 1A, maize protoplast transfection with 40% PEG in 0.4 M mannitol resulted in better expression of GFP compared to those with 0.2 M mannitol. As would be expected, an increase in incubation time resulted in higher GFP expression regardless of the concentration of mannitol.
Maize protoplasts were transfected with constructs carrying GFP driven by two promoters commonly used for high-level expression in maize: the cauliflower mosaic virus (CMV) 35S promoter or the maize ubiquitin promoter. According to microscopic observation (Figure 1B) and western blot analysis (Figure 1C), expression of GFP was stronger when driven by the maize ubiquitin promoter than that by the CMV 35S promoter. Further tests with protoplasts isolated from Early Sunglow and Silver Queen hybrids revealed their remarkable longevity after transfection with pCXUN-HA-GFP. As shown in Figure 1D, GFP expression was detected in both hybrids four days post transfection (dpt). Based on these observations, we adopted 0.4 M mannitol for protoplast transfection and utilized the ubiquitin promoter in subsequent CRISPRa and CRISPRi vector construction. By comparing protoplasts with GFP signal to those without in a given area, we observed a transformation efficiency range of 60-70% which is similar to that reported for rice protoplasts .
Expression of dCas9 variants in planta
To develop the maize CRISPRi and CRISPRa toolkit, we assembled the following series of constructs using a pTF101.1rev binary vector backbone: pDA2 (conferring dCas9 expression), pDA3 (dCas9-VP64, conferring dCas9-mediated expression activation), pDA4 (dCas9-SRDX, conferring dCas9-mediated expression repression), and pDA5 (dCas9-TV, conferring stronger dCas9-mediated expression activation). Each dCas9 derivative is N-terminally Flag-tagged and driven by the maize ubiquitin promoter (Additional File 2). Also included is a dual 35S-driven BAR cassette to confer glufosinate resistance. To confirm the expression of dCas9 variants in planta within whole plants, we infiltrated agrobacteria strains carrying different pDA vectors into Nicotiana benthamiana leaves. Three days after infiltration, total protein was isolated and expression of dCas9 variants was detected at the expected size using an anti-Flag antibody (Figure 2). To confirm expression in maize plants by protoplast transient expression, we cloned the HindIII and SbfI fragment containing the dCas9 expression cassette from each pDA construct into the HindIII and PstI sites of pXUN, thereby decreasing the size of the construct by about 7 kb to increase the protoplast transfection rate. Total protein was isolated 16 h after maize protoplast transfection, and expression of the dCas9 variants were detected at the expected size by western blot with the anti-Flag antibody (Figure 2). We observed that pDA2 showed higher expression compared to the pDA3 and pDA4 dCas9 variants in both N. benthamiana plants and maize protoplasts. While the reason is not clear, we speculate that the addition of activation and repression domains to the dCas9 construct leads to this expression reduction. Although a direct comparison to dCas9 was not included, Li, et al. (2017) observed a similar trend with western blot analysis of dCas9-activation constructs in Arabidopsis protoplasts, where increases in the overall size of the activation domains correlated with reduced expression.
Testing the transcriptional changes of ChlH and TrxH using CRISPR/dCas9 constructs in protoplasts
To determine the effectiveness of the pDA vectors for either CRISPRa or CRISPRi, we designed gRNAs targeting the promoter of the maize Subunit H of magnesium chelatase gene (ChlH), a marker gene whose mutation in whole plants leads to yellowing seedling phenotype due to defects in chloroplast development . Four gRNAs targeting different regions of the ChlH promoter (Additional File 1) were designed and co-expressed with pDA2 (dCas9) or pDA4 (dCas9-SRDX) in protoplasts. qRT-PCR analysis showed that gRNA1, gRNA2, and gRNA3 co-transfection with pDA2 resulted in an approximate 25% reduction in ChlH expression, while gRNA4 had no effect (Figure 3A). Co-expression of pDA4 with gRNA2 or gRNA4 resulted in nearly a 75% or 50% reduction in ChlH expression, respectively, compared to the negative control (Figure 3B). These data show that while dCas9 has some transcription repression activity, this can be enhanced with the addition of the SRDX suppressor. For CRISPRa, we analyzed Thioredoxin H (TrxH), a gene whose increased transcription in whole plants confers resistance to sugarcane mosaic virus (SCMV) (Liu et al., 2017). As with ChlH, we tested four gRNAs targeting the TrxH promoter (Additional File 1). When co-expressed with pDA3 (dCas9-VP64), gRNA2 or gRNA4 resulted in about a two-fold increase in TrxH transcripts (Figure 3C). These two analyses confirmed that our pDA3 and pDA4 vectors can be used for CRISPRa and CRISPRi approaches, respectively, and gRNAs for target genes can be tested in maize protoplasts for further experiments.
Testing the transcriptional changes of PDS1 using three CRISPR/dCas9 constructs in maize protoplasts
To demonstrate CRISPRi with multiplexed constructs, gRNAs targeting the promoter of the maize phytoene desaturase1 (PDS1) gene were designed. PDS1 is a commonly used marker gene for virus-induced gene silencing (VIGS) as well as CRISPR/Cas9 genome editing analysis across a range of plant species, where silencing or mutation of the gene culminates in an easily observed photobleaching phenotype [32–36]. To test PDS1 expression, we designed four gRNAs targeting the PDS1 promoter (Additional File 1). Next, to multiplex two gRNAs in the same construct, we added three tRNAs to flank each side of the gRNA-scaffold sequences. The addition of tRNAs for multiplexed constructs is a widely-used method to create individual, discrete gRNAs in vivo from a single construct by the activity of the plant’s endogenous tRNA-processing machinery [37–39]. Multiple combinations of these four PDS1 gRNAs were then co-transfected with either pDA2, pDA3, or pDA4 into maize protoplasts. We determined that a combination of gRNA2 and gRNA3 co-transfected with pDA4 showed a decrease of about 60% in PDS1 transcription compared to the negative control, as measured by qRT-PCR (Figure 3D). We also tested whether the PDS1 gRNAs could be used for transcription activation with pDA3 (dCas9-VP64) in maize protoplasts. With pDA3, a combination with gRNA2 and gRNA3 showed about 2.5 times of PDS1 transcription activation (Figure 3E).
The dual-luciferase assay is rapid, sensitive, and reliable for analysis of transcriptional repressors, gene expression or functional interaction of signaling molecules . The 35S-driven Renilla luciferase serves as an internal and transfection control, while Firefly luciferase is driven by a promoter of interest . We tested whether this assay could be utilized to assess the repression or activation of PDS1 using our CRISPR/dCas9 constructs. The vectors for this system were generated by first cloning a 1.4 kb promoter fragment of PDS1 into the BamHI site of pGreenII-800-RNA1-Luc, resulting in the construct pGreenII-800-RNA1-PDS1:Luc (Additional File 3). Next, PDS1 gRNAs were cloned into the BtgZI and BsaI sites with the different multiplex combinations as described above. After the co-transfection of PDS1 gRNAs in pGreenII-800-RNA1-PDS1:Luc with CRISPR/dCas9 constructs, the protoplasts were lysed for Firefly and Renilla luciferase activity according to manufacturer’s instructions (Promega). As shown in the preliminary results of Additional File 3, the relative expression patterns of PDS1 measured by the dual-luciferase assay were similar to that detected by qRT-PCR (Figure 3D-E). Overall, a combination of gRNA2 and gRNA3 showed the best activation and suppression of PDS1 with pDA3 and pDA4, respectively. As an additional control for the protoplast transfection rate, we modified the pGreenII-800-RNAi-Luc vector by inserting a GFP-expression cassette. Prior to gRNA analysis via dual-luciferase or qRT-PCR, the transfection rate with GreenII-800-RNAI-GFP-Luc can be determined by western blot analysis (Additional File 3).