Supplemental Information
Robust transcriptional activation in plants using multiplexed CRISPR-Act2.0 and mTALE-Act systems
Levi G. Lowder1,7, Jianping Zhou2,7, Yingxiao Zhang3, Aimee Malzahn3, Zhaohui Zhong2Tzung-Fu Hsieh4, Daniel F. Voytas5, Yong Zhang2*, Yiping Qi1,3,6*
1Department of Biology, East Carolina University, Greenville, North Carolina 27858, USA;
2Department of Biotechnology, School of Life Science and Technology,Center for Informational Biology,University of Electronic Science and Technology of China,Chengdu610054,China
3Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA;
4Department of Plant and Microbial Biology &Plants for Human Health Institute,North Carolina State University, North Carolina Research Campus, Kannapolis,NC 28081, USA;
5Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA;
6Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850, USA.
7These authors contributed equally to this work.
* Corresponding authors: Yiping Qi ()
Yong Zhang ()
INDEX
SUPPLEMENTAL METHODS
Assembly of a CRISPR-Act2.0 T-DNA vector with triplex gRNAs……..…….…..page 3-4
Assembly of an mTALE-Act T-DNA vector with triplex TALEs……….…………..page 5
List of the 26 T-DNA vectors used for plant transformation………………………page6
Details on construction of all the vectors in this study………………………....…page 7-12
Sequence and annotation of target genes and target sites………………….……page 13-18
List of oligos and gBlocks used in this study………………………………………..page 19-24
References…………………………………………………………………………………..page 24
SUPPLEMENTAL Figures…………………………………….………………….page 25-31
Assembly of a CRISPR-Act2.0 T-DNA vector with triplex gRNAs
Step1. Cloning guide RNA (gRNA) into gRNA2.0 expression vectors
- Linearize guide RNA expression plasmids (pYPQ131A/B/C/D2.0, 132A/B/C/D2.0 and 133A/B/C/D2.0; pYPQ141A/B/C/D2.0 for single gRNA)
1. First digestion with BglII and SalI (Optional but recommended for zero-background cloning)
H2O 14 μl
gRNA plasmid (~100 ng/μl) 20 μl
10X NEB buffer 3.1 4 μl
BglII (10 u/μl; NEB) 1 μl
SalI-HF (10 u/ μl; NEB) 1 μl
Total 40 μl
37 0C, 3 hrs
2. Second digestion with Esp3I (BsmBI)
Purify 1st digestion products using Qiagen PCR purification kit, elute DNA with 35 μl ddH2O, set up digestion reaction as follow:
Digested gRNA plasmid (from step 1)32 μl
10X OPTIZYME buffer 44 μl
DTT (20 mM)2 μl
EPS3I (10 u/μl; Thermo Scientific)2 μl
Total40 μl
37 0C, O/N
Inactivate enzymes at 800C denature for 20 min, purify the vector using Qiagen PCR purification kit, and quantify DNA concentration using Nanodrop.
- Cloning Oligos into linearized pYPQ13N-2.0 vector
3. Oligo phosphorylation and annealing
sgRNA oligo forward (100 μM)1 μl
sgRNA oligo reverse (100 μM)1 μl
10X T4 Polynucleotide Kinase buffer1 μl
T4 Polynucleotide Kinase (10 u/μl; NEB) 0.5 μl
ddH2O6.5 μl
Total 10 μl
Phosphorylate and anneal the oligos using 37 0C for 30 min; 95 0C for 5 min; ramp down to 25 0C at 5 0C min-1 (i.e., 0.08 0C/second) using a thermocycler (alternatively: cool down in boiled water) .
4. Ligate oligos into linearized gRNA expression vector and transformation of E.coli DH5α cells
ddH2O 6.5 ul
10X NEB T4 ligase buffer 1 μl
Linearized gRNA2.0 plasmid1 μl
Diluted annealed Oligos (1:200 dilution)1 μl
T4 ligase0.5 μl
Total10 μl
@RT for 1hr
5. Transform E.coli DH5α cells and plate transformed cells on a Tet+ (5ng/ul) LB plate; 37 0C, O/N
6. Mini-prep two independent clones and verify gRNAs by Sanger sequencing with primer pTC14-F2 (for pYPQ131, 132 and 133 based vectors) or M13-F (for pYPQ141 based vectors).
Step2. Golden Gate Assembly of 3 gRNA2.0 cassettes
- Set up Golden Gate reaction following this example:
Assembly of 3 guide RNAs
H2O 4 µl
10X T4 DNA ligase buffer (NEB) 1 µl
pYPQ143 (100 ng/ µl) 1 µl
pYPQ131-gRNA1 (100 ng/ µl) 1 µl
pYPQ132-gRNA2 (100 ng/ µl) 1 µl
pYPQ133-gRNA3 (100 ng/ µl) 1 µl
BsaI (NEB) 0.5 µl
T4 DNA ligase (NEB) 0.5 µl
Total 10 µl
- Run Golden Gate program in a thermocycler as follows:
37 0C, 5 min
16 0C, 10 min
50 0C, 5 min
80 0C, 5 min
Hold at 10 0C
- Transform E. coli DH5α cells and plate transformed cells onto a Spe+ (100 µg/ml) LB plate. [Optional: Blue-white screen can be applied because guide RNA expression cassettes will replace the LacZ gene in pYPQ143 recipient plasmid]
- Mini-prep two independent clones and verify by restriction digestion
Step 3. Gateway Assembly of Multiplex CRISPR-Cas9 system into a binary vector
- Set up Gateway LR reaction as following:
Cas9 entry vector pYPQ173 (25ng/ µl) 2 μl
Guide RNA entry vector (25ng/ µl) 2 μl
Destination vector (100 ng/ µl) 2 μl
LR Clonase II 1 μl
Total 7 μl
@RT for 1hr (O/N recommended)
- Transform E. coli DH5α cells and plate transformed cells on a Kan+ (50 µg/ml) LB plate
- Mini-prep two independent clones and verify by restriction digestion
Assembly of an mTALE-Act T-DNA vector with triplex TALEs
The overall assembly is based on our previously published protocol (Cermak et al., 2011). However, we noticed that assembly of 10 TALE repeats into pFUS_A vector is not very efficient. To improve the assembly efficiency, we reduced pFUS-A capacity to accommodate 8 TALE repeats by generating a new pFUS_A8 vector. This vector was used to assemble the first 8 TALE repeats with Golden Gate method.
Briefly, the first 8 TALE repeats (highlighted in blue in Table S1) and the remaining repeats (highlighted in red in Table S1) were assembled into pFUS_A8 and pFUS_B vectors respectively with a first-round Golden Gate reaction based on BsaI. The assembled repeats were sequence-confirmed with primers pCR8-F1 and pCR8-R1. Then, these repeats were put together into pZHY500(Zhang et al., 2013) with a second-round Golden Gate reaction based on Esp3I (or BsmBI). The assembled full TALE repeats were then excised from XbaI and BamHI sites and cloned into pYPQ121 or pYPQ127B at XbaI and BamHI sites, or at NheI and BglII sites depending on the TALE position. Finally, the T-DNA transformation vector was generated with a Gateway reaction with attL1-attR5 pYPQ121 (that contains two TALE-VP64), attL5-attL2 pYPQ127B (that contains an additional TALE-VP64) and an attR1-attR2 destination vector (pYPQ202 in this study)(Tang et al., 2017).
If two or less TALE-VP64 genes will be added to the assembly, pYPQ140 (which is an attL5-attL2 empty vector) will be used, in replacement of pYPQ127B.
Table S1 that summarizes the TALE assembly information
Note RVD correspondence: NG for T, NN for G, NI for A, HD for C.
List of the 26 T-DNA vectors used for plant transformation
Table S2.
# / Name of T DNA construct / Corresponding data21 / pYPQ202-dCas9-VP64- PAP1-gR1,gR2,gR3 / Figure 2B
25 / pYPQ202-dCas9-VP64- FIS2-gR1,gR2,gR3 / Figure 2C
94 / pYPQ202-TALE-VP64-RBP-CSTF-GL1-11,5,7 / Figure 4B-D; Supplemental Fig 3C; Supplemental Fig 4A-C
95 / pYPQ202-TALE-VP64-RBP-CSTF-GL1-12,6,8 / Figure 4F-H; Supplemental Fig 3D; Supplemental Figure 4D-F
123 / pYPQ202-dCas9-VP64-T2A-VP64-MS2-VP64-miR319-gR2 / Supplemental Fig 6A
127 / pYPQ202-dCas9-VP64-T2A-MS2-VP64-miR319-gR1,gR2,gR3 / Supplemental Fig 6B
128 / pYPQ202-dCas9-VP64-T2A-MS2-VP64-PAP1-gR1,gR2,gR3 / Figure 2K
130 / pYPQ202-dCas9-VP64-T2A-MS2-EDLL-PAP1-gR1,gR2,gR3 / Figure 2H
194 / pMDC99-GUS-Intron-TALEA-11-5-7 / Supplemental Fig 3C
195 / pMDC99-GUS-Intron-TALEA-12-6-8 / Supplemental Fig 3D
196 / pYPQ202-dCas9-VP64-EDLL-FIS2-gR1,gR2,gR3 / Figure 2F
198 / pYPQ202-dCas9-VP64-EDLL-PAP1-gR1,gR2,gR3 / Figure 2E
247 / pMDC-pMiR319-dCas9-VP64-T2A-MS2-VP64-miR319-gR1, gR2, gR3 / Figure 5C
258 / pYPQ202-TALE-VP64-PAP1-37,38 / Figure 3B
259 / pYPQ202-TALE-VP64-miR319-39-40 / Supplemental Fig 6C
260 / pMDC-pPAP1-TALE-VP64-PAP1-37,38 / Figure 5E
261 / pMDC-pMiR319-TALE-VP64-miR319-39-40 / Figure 5D
313 / pYPQ202-dCas9-VP64-T2A-MS2-VP64-FIS2-gR1,gR2,gR3 / Figure 2L
315 / pYPQ202-dCas9-VP64-T2A-MS2-VP64-UCL1-gR1,gR2,gR3 / Supplemental Fig 2
317 / pYPQ202-dCas9-VP64-T2A-MS2-EDLL-FIS2-gR1,gR2,gR3 / Figure 2I
585 / pYPQ203-dCas9-VP64-N21-23-25 / Figure 3C
586 / pYPQ203-dCas9-VP64-T2A-MS2-VP64-N21-23-25 / Figure 3C
589 / pYPQ203-dCas9-VP64-N21-22 / Figure 3A
590 / pYPQ203-dCas9-VP64-T2A-MS2-VP64N21-22 / Figure 3A
591 / pYPQ203-dCas9-VP64-N23-24 / Figure 3B
592 / pYPQ203-dCas9-VP64-T2A-MS2-VP64-N23-24 / Figure 3B
Details on construction of the vectors in this study
Note: All the restriction enzymes, T4 ligase and T4 Polynucleotide kinase were purchased from NEB. Esp3I (BsmBI) is from Thermo Scientific.The miniprep kit is from Midwest Scientific, and the gel extraction kit and PCR purification kit are from Qiagen. Synthetic DNA oligos and genes (gBlocks) are from Integrated DNA Technologies (IDT).
- Construction of vectors in the CRISPR-Act2.0 toolkit:
Construction of pYPQ141A, pYPQ141B, pYPQ141C and pYPQ141D:
Part of gRNA2.0 cassette was PCR amplified from MS2-gR2.0 gBlock with primers gRNA2.0-F and gRNA2.0-R. The ~170bp PCR product was cloned into pYPQ141A, pYPQ141B, pPYQ141C and pYPQ141D at BglII and BanI sites to generate pYPQ141A2.0, pYPQ141B2.0, pYPQ141C2.0 and pYPQ141D2.0 respectively.
Construction of pYPQ131A2.0, pYPQ131B2.0, pYPQ131C2.0, pYPQ131D2.0, pYPQ132A2.0, pYPQ132B2.0, pYPQ132C2.0, pYPQ132D2.0, pYPQ133A2.0, pYPQ133B2.0, pYPQ133C2.0, and pYPQ133D:
The gRNA2.0 expression cassettes were PCR amplified from pYPQ141A2.0 with primerspYPQ131-F and pYPQ131R, from pYPQ141B2.0 with primers pYPQ131B-F and pYPQ131B-R, from pYPQ141C2.0 with primers pYPQ131C-F and pYPQ131C/D-R, from pYPQ141D2.0 with primers pYPQ131D-F and pYPQ131C/D-R. The PCR products were then cloned into vector pHD1(Cermak et al., 2011) at the two BsaI sites, resulting in pYPQ131A2.0, pYPQ131B2.0, pYPQ131C2.0 and pYPQ131D2.0 respectively. The gRNA2.0 expression cassettes were PCR amplified from pYPQ141A2.0 with primers pYPQ132-F and pYPQ132-R, from pYPQ141B2.0 with primers pYPQ132B-F and pYPQ132B-R, from pYPQ141C2.0 with primers pYPQ132C-F and pYPQ131C/D-R, from pYPQ141D2.0 with primers pYPQ132D-F and pYPQ132C/D-R. The PCR products were cloned into pHD2 (Cermak et al., 2011)at BsaI sites to make pYPQ132A2.0, pYPQ132B2.0, pYPQ132C2.0 and pYPQD2.0. Similarly, pYPQ133A2.0, pYPQ133B2.0, pYPQ133C2.0 and pYPQ133D2.0 were made by cloning PCR ampliconsinto pHD3 (Cermak et al., 2011) at BsaI sites. These amplicons were from pYPQ141A2.0 with primer pYPQ133-F and pYPQ133-R, from pYPQ141B2.0 with primers pYPQ133B-F and pYPQ133B-R, from pYPQ141C2.0 with primers pYPQ133C-F and pYPQ133C/D-R, and lastly from pYPQ141D2.0 with primers pYPQ133D-F and pYPQ133C/D-R.
Construction of pYPQ174:
T2A-MS2-NLS-EDLL was PCR amplified from MS2-gR2.0 gBlock with primers VP64-T2A-F and EDLL-Nos-R. The PCR product was mixed with AatII-linearized pYPQ152 vector (Lowder et al., 2015) for homologous recombination in E.coli, which resulted in pYPQ174.
Construction of pYPQ171:
EDLL was PCR amplified from MS2-gR2.0 gBlock with primers 5XGS-EDLL-F and 5XGS-EDLL-R. The PCR product was mixed with AatII-linearized pYPQ152 vector (Lowder et al., 2015) for homologous recombination in E.coli, which resulted in pYPQ171.
Construction of pYPQ173:
VP64 sequence was PCR-amplified from pYPQ152 using primers KpnI-VP64-F and VP64-HindIII-R. The PCR product was cloned into pYPQ174 at KpnI and HindIII sites to generate pYPQ173.
- Construction of vectors in the mTALE-Act toolkit:
Construction of pYPQ121:
pYPQ121 was generated by sequential cloning of two gBlocks (GB-YPQ1 and GB-YPQ3) into pYPQ152 by replacing dCas9-VP64 at NcoI and AatII sites.
Construction of pYPQ127B:
First, pYPQ127 was generated by sequential cloning of two gBlocks (GB-YPQ1 and GB-YPQ2) into pYPQ141A at NcoI and SpeI sites. Then,the 2X35S promoter was amplified from pMDC32 using primers 35S-F (HindIII) and 35S-R (KpnI) and cloned into HindIII and KpnI sites of pYPQ127 to make pYPQ127B.
- Construction of pFUS-A8
pFUS_A8 was generated by ligating the linker A8 (by annealing A8-LNK-F and A8-LNK-R) into AgeI and NcoI sites of pFUS_A (Cermak et al., 2011).
- Construction of 26 T-DNA vectors:
Construction of Vector #21 (pYPQ202-dCas9-VP64-PAP1-gR1,2,3) and #25 (pYPQ202-dCas9-VP64-FIS2-gR1:
These two vectors were previously made (Lowder et al., 2015).
Construction of Vector #94 (pYPQ202-TALE-VP64-RBP-CSTF-GL1-11,5,7):
First, Golden Gate assembled TALE repeats #11 (for targeting RBP-DR1 promoter; Table S1) was excised from pZHY500-11 with XbaI and BamHI and cloned into pYPQ121 at XbaI and BamHI sites, to make pYPQ121-11. Then,assembled TALE repeats #5 (for targetingCSTF64 promoter, Table S1) was excised from pZHY500-5 and cloned into pYPQ121-11 at NheI and BglII sites to make pYPQ121-11&5. Meanwhile, assembled TALE repeats #7 (for targeting GL1 promoter, Table S1) was excised from pZHY500-7 with XbaI and BamHI and cloned into the same sites of pYPQ127-B to make pYPQ127B-7. The final T-DNA vector was generated by a MultiSite Gateway recombination with pYPQ121-11&5, pYPQ127B-7, and pYPQ202.
Construction of Vector #95 (pYPQ202-TALE-VP64-RBP-CSTF-GL1-12,6,8):
First, Golden Gate assembled TALE repeats #12 (for targeting RBP-DR1 promoter; Table S1) was excised from pZHY500-12 with XbaI and BamHI and cloned into pYPQ121 at XbaI and BamHI sites, to make pYPQ121-12. Then, assembled TALE repeats #5 (for targeting CSTF64 promoter, Table S1) was excised from pZHY500-6 and cloned into pYPQ121-12 at NheI and BglII sites to make pYPQ121-12&6. Meanwhile, assembled TALE repeats #8 (for targeting GL1 promoter, Table S1) was excised from pZHY500-8 with XbaI and BamHI and cloned into the same sites of pYPQ127-B to make pYPQ127B-8. The final T-DNA vector was generated by a MultiSite Gateway recombination with pYPQ121-12&6, pYPQ127B-8, and pYPQ202.
Construction of Vector #123 (pYPQ202-dCas9-VP64-T2A-VP64-MS2-VP64-miR319-gR2):
First, oligos corresponding to miR319-gR2 (miR319-gR2-top and miR319-gR2-bottom) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ141A through ligation, resulting in pYPQ141A-miR319-gR2. This gRNA entry clone was then mixed with pYPQ173 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-dCas9-VP64-T2A-VP64-MS2-VP64-miR319-gR2.
Construction of Vector #127 (pYPQ202-dCas9-VP64-T2A-MS2-VP64-miR319-gR1,gR2,gR3):
First, oligos corresponding to miR319-gR1 (miR319-gR1-top and miR319-gR1-bottom), miR319-gR2 (miR319-gR2-top and miR319-gR2-bottom) and miR319-gR3 (miR319-gR3-top and miR319-gR3-bottom) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A2.0, pYPQ132A2.0 and pYPQ133A2.0 respectively (step 1). These three vectors were then mixed with pYPQ143 for a Golden Gate reaction to make pYPQ143-miR319-gRNA2.0-1,2,3 (step 2). Finally, this gRNA entry clone was mixed with pYPQ173 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-dCas9-VP64-T2A-MS2-VP64-miR319-gR1,gR2,gR3 (step 3).
Construction of Vector #128 (pYPQ202-dCas9-VP64-T2A-MS2-VP64-PAP1-gR1,gR2,gR3):
First, oligos corresponding to PAP1-gR1 (PAP1-gR1-top and PAP1-gR1-bottom), PAP1-gR2 (PAP1-gR2-top and PAP1-gR2-bottom) and PAP1-gR3 (PAP1-gR3-top and miR319-gR3-bottom) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A2.0, pYPQ132A2.0 and pYPQ133A2.0 respectively (step 1). These three vectors were then mixed with pYPQ143 for a Golden Gate reaction to make pYPQ143- PAP1-gRNA2.0-1,2,3 (step 2). Finally, this gRNA entry clone was mixed with pYPQ173 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-dCas9-VP64-T2A-MS2-VP64- PAP1-gR1,gR2,gR3 (step 3).
Construction of Vector #130 (pYPQ202-dCas9-VP64-T2A-MS2-EDLL-PAP1-gR1,gR2,gR3):
First, oligos corresponding to PAP1-gR1 (PAP1-gR1-top and PAP1-gR1-bottom), PAP1-gR2 (PAP1-gR2-top and PAP1-gR2-bottom) and PAP1-gR3 (PAP1-gR3-top and miR319-gR3-bottom) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A2.0, pYPQ132A2.0 and pYPQ133A2.0 respectively (step 1). These three vectors were then mixed with pYPQ143 for a Golden Gate reaction to make pYPQ143- PAP1-gRNA2.0-1,2,3 (step 2). Finally, this gRNA entry clone was mixed with pYPQ174 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-dCas9-VP64-T2A-MS2-EDLL- PAP1-gR1,gR2,gR3 (step 3).
Construction of minimal promoter reporter T-DNA vectors #194 (pMDC99-GUS-Intron-TALEA-11-5-7) and 195 (pMDC99-GUS-Intron-TALEA-12-6-8):
First, a pair of oligos (TALE-11-5-7-F and TALE-11-5-7-R) containing the corresponding three TALE target sites were phosphorylated, annealed and then ligated into BamHI and SpeI sites of pCR8-GUS-Intron-GExp vector (Lowder et al., 2015), generating pCR8-GUS-Intron-TALEA-11-5-7. Then, the reporter T-DNA vector #194 was made by a Gateway recombination between pCR8-GUS-Intron-TALEA-11-5-7 and pMDC99 (Curtis and Grossniklaus, 2003). The T-DNA vector #195 was made in a similar way using oligos TALE-12-6-8-F and TALE-12-6-8R.
Construction of Vector #196 (pYPQ202-dCas9-VP64 -EDLL-FIS2-gR1,gR2,gR3):
First, oligos corresponding to FIS2-gR1 (FIS2-gR1-top and FIS2-gR1-bottom), FIS2-gR2 (FIS2-gR2-top and FIS2-gR2-bottom) and FIS2-gR3 (FIS2-gR3-top and FIS2-gR3-bottom) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A, pYPQ132A and pYPQ133A2 respectively (step 1). These three vectors were then mixed with pYPQ143 for Golden Gate reaction to make pYPQ143-FIS2-gR-1,2,3 (step 2). Finally, this gRNA entry clone was mixed with pYPQ171 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-dCas9-VP64-EDLL- FIS2-gR1,gR2,gR3 (step 3).
Construction of Vector #198 (pYPQ202-dCas9-VP64 -EDLL-PAP1-gR1,gR2,gR3):
First, oligos corresponding to PAP1-gR1 (PAP1-gR1-top and PAP1-gR1-bottom), PAP1-gR2 (PAP1-gR2-top and PAP1-gR2-bottom) and PAP1-gR3 (PAP1-gR3-top and PAP1-gR3-bottom) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A, pYPQ132A and pYPQ133A2 respectively (step 1). These three vectors were then mixed with pYPQ143 for a Golden Gate reaction to make pYPQ143- PAP1-gR-1,2,3 (step 2). Finally, this gRNA entry clone was mixed with pYPQ171 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-dCas9-VP64-EDLL- PAP1-gR1,gR2,gR3 (step 3).
Construction of Vector #247 (pMDC-pMiR319-dCas9-VP64-T2A-MS2-VP64-miR319-gR1, gR2, gR3):
First, a 1.1kb miR319 promoter was PCR amplified from Arabidopsis Columbia (Col) DNA by following a nested PCR procedure where two sets of primers were used: pmiR319-Nest-F and pmiR319-Nest-R for the first round of PCR, and pmiR319-F-SbfI and pmiR319-R-KpnI for the second round of PCR. Then, the PCR product was cloned into pMDC32 to replace the 2X35S promoter at SbfI and KpnI sites, to generate the new destination vector pMDC-pMiR319. Finally, the gRNA entry clone pYPQ143-miR319-gRNA2.0-1,2,3 generated earlier was mixed with pYPQ173 and pMDC-pMiR319in a MultiSiteGateway reaction to make pYPQ202-dCas9-VP64-T2A-MS2-VP64-miR319-gR1,gR2,gR3 (step 3).
Construction of Vector #258 (pYPQ202-TALE-VP64-PAP1-37,38):
First, Golden Gate assembled TALE repeats #37 and #38 (both targeting RBP-DR1 promoter; Table S1) were excised from pZHY500-37 and pZHY500-38 with XbaI and BamHI and sequentially cloned into pYPQ121 at XbaI-BamHI sites, and NheI-BglII sites respectively to make pYPQ121-37&38. Then, the final T-DNA vector was generated by a MultiSite Gateway recombination reaction with pYPQ121-37&38, pYPQ140 (empty attL5-attL2 vector), and pYPQ202.
Construction of Vector #259 (pYPQ202-TALE-VP64-miR319-39,40):
First, Golden Gate assembled TALE repeats #39 and #40 (both targeting miR319 promoter; Table S1) were excised from pZHY500-39 and pZHY500-40 with XbaI and BamHI and sequentially cloned into pYPQ121 at XbaI-BamHI sites, and NheI-BglII sites respectively to make pYPQ121-39&40. Then, the final T-DNA vector was generated by a MultiSite Gateway recombination reaction with pYPQ121-39&40, pYPQ140 (empty attL5-attL2 vector), and pYPQ202.
Construction of Vector #260 (pMDC-pPAP1-TALE-VP64-PAP1-37,38):
First, the 1.2kb PAP1 promoter was PCR amplified from Col DNA by following a nested PCR procedure where two sets of primers were used: PAP1-Nest-F and PAP1-Nest-R for the first round of PCR, and PAP1-F-SbfI and PAP1-R-KpnI for the second round of PCR. Then, the PCR product was cloned into pMDC32 to replace the 2X35S promoter at SbfI and KpnI sites, to generate the new destination vector pMDC-PAP1.The final T-DNA vector was generated by a MultiSite Gateway recombination reaction with pYPQ121-37&38, pYPQ140 (empty attL5-attL2 vector), and pMDC-PAP1.
Construction of Vector #261 (pMDC-pMiR319-TALE-VP64-miR319-39-40):
The final T-DNA vector was generated by a MultiSite Gateway recombination reaction with pYPQ121-39&40, pYPQ140 (empty attL5-attL2 vector), and pMDC-pMiR319.
Construction of Vector #313 (pYPQ202-dCas9-VP64-T2A-MS2-VP64-FIS2-gR1,gR2,gR3):
First, oligos corresponding to FIS2-gR1 (FIS2-gR1-top and FIS2-gR1-bottom), FIS2-gR2 (FIS2-gR2-top and FIS2-gR2-bottom) and FIS2-gR3 (FIS2-gR3-top and FIS2-gR3-bottom) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A2.0, pYPQ132A2.0 and pYPQ133A2.0 respectively (step 1). These three vectors were then mixed with pYPQ143 in a Golden Gate reaction to make pYPQ143-FIS2-gRNA2.0-1,2,3 (step 2). Finally, this gRNA entry clone was mixed with pYPQ173 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-dCas9-VP64-T2A-MS2-VP64- FIS2-gR1,gR2,gR3 (step 3).
Construction of Vector #315 (pYPQ202-dCas9-VP64-T2A-MS2-VP64-UCL1-gR1,gR2,gR3):
First, oligos corresponding to UCL1-gR1 (UCL1-gR1-F and UCL1-gR1-R), UCL1-gR2 (UCL1-gR2-F and UCL1-gR2-R) and UCL1-gR3 (UCL1-gR3-F and UCL1-gR3-R) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A2.0, pYPQ132A2.0 and pYPQ133A2.0 respectively (step 1). These three vectors were then mixed with pYPQ143 in a Golden Gate reaction to make pYPQ143- UCL1-gRNA2.0-1,2,3 (step 2). Finally, this gRNA entry clone was mixed with pYPQ173 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-dCas9-VP64-T2A-MS2-VP64- UCL1-gR1,gR2,gR3 (step 3).
Construction of Vector #317 (pYPQ202-dCas9-VP64-T2A-MS2-EDLL-FIS2-gR1,gR2,gR3):
The T-DNA vector was generated with a MultiSite Gateway reaction using pYPQ143-FIS2-gRNA2.0-1,2,3, pYPQ174 and pYPQ202.
Construction of Vector #585 (pYPQ203-dCas9-VP64-N21-23-25):
First, oligos corresponding to Os03g01240-gR1 (N21), Os04g39780-gR1 (N23) and Os11g35410-gR1 (N25) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A, pYPQ132A and pYPQ133A respectively (step 1). These three vectors were then mixed with pYPQ143 in a Golden Gate reaction to make pYPQ143-N21-23-25 (step 2). Finally, this gRNA entry clone was mixed with pYPQ152 and pYPQ203 in a MultiSite Gateway reaction to make pYPQ203-dCas9-VP64-N21-23-25 (step 3).
Construction of Vector #586 (pYPQ203-dCas9-VP64-T2A-MS2-VP64-N21-23-35):
First, oligos corresponding to Os03g01240-gR1 (N21), Os04g39780-gR1 (N23) and Os11g35410-gR1 (N25) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A2.0, pYPQ132A2.0 and pYPQ133A2.0 respectively (step 1). These three vectors were then mixed with pYPQ143 in a Golden Gate reaction to make pYPQ143-gRNA2.0-N21-23-25 (step 2). Finally, this gRNA entry clone was mixed with pYPQ173 and pYPQ203 in a MultiSite Gateway reaction to make pYPQ203-dCas9-VP64-T2A-MS2-VP64-N21-23-25(step 3).
Construction of Vector #589 (pYPQ203-dCas9-VP64-N21-22):
First, oligos corresponding to Os03g01240-gR1 (N21) andOs03g01240-gR2 (N22) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A and pYPQ132A respectively (step 1). These two vectors were then mixed with pYPQ142in a Golden Gate reaction to make pYPQ142-N21-22(step 2). Finally, this gRNA entry clone was mixed with pYPQ152 and pYPQ203 in a MultiSite Gateway reaction to make pYPQ203-dCas9-VP64-N21-22 (step 3).
Construction of Vector #590 (pYPQ203-dCas9-VP64-T2A-MS2-VP64-N21-22):
First, oligos corresponding to Os03g01240-gR1 (N21) andOs03g01240-gR2 (N22) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A2.0 and pYPQ132A2.0 respectively (step 1). These two vectors were then mixed with pYPQ142in a Golden Gate reaction to make pYPQ142-gRNA2.0-N21-22 (step 2). Finally, this gRNA entry clone was mixed with pYPQ173 and pYPQ203 in a MultiSite Gateway reaction to make pYPQ203-dCas9-VP64-T2A-MS2-VP64-N21-22 (step 3).
Construction of Vector #591 (pYPQ203-dCas9-VP64-N23-24):
First, oligos corresponding to Os04g39780-gR1 (N23) andOs04g39780-gR2 (N24) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A and pYPQ132A respectively (step 1). These two vectors were then mixed with pYPQ142in a Golden Gate reaction to make pYPQ142-N23-24 (step 2). Finally, this gRNA entry clone was mixed with pYPQ152 and pYPQ203 in a MultiSite Gateway reaction to make pYPQ203-dCas9-VP64-N23-24 (step 3).
Construction of Vector #592 (pYPQ203-dCas9-VP64-T2A-MS2-VP64-N23-24):
First, oligos corresponding to Os04g39780-gR1 (N23) andOs04g39780-gR2 (N24) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A2.0 and pYPQ132A2.0 respectively (step 1). These two vectors were then mixed with pYPQ142in a Golden Gate reaction to make pYPQ142-gRNA2.0-N23-24 (step 2). Finally, this gRNA entry clone was mixed with pYPQ173 and pYPQ203 in a MultiSite Gateway reaction to make pYPQ203-dCas9-VP64-T2A-MS2-VP64-N23-24 (step 3).