Volume 11, Issue 2, Pages 245-256 (February 2018) Robust Transcriptional Activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems  Levi G. Lowder, Jianping Zhou, Yingxiao Zhang, Aimee Malzahn, Zhaohui Zhong, Tzung-Fu Hsieh, Daniel F. Voytas, Yong Zhang, Yiping Qi  Molecular Plant  Volume 11, Issue 2, Pages 245-256 (February 2018) DOI: 10.1016/j.molp.2017.11.010 Copyright © 2017 The Author Terms and Conditions

Figure 1 Gateway and Golden Gate Entry Vectors for Assembly of Multiplexed CRISPR-Act2.0 and mTALE-Act Systems. (A and B) Multiplex CRISPR dCas9 activation entry vectors. (A) pYPQ173 Gateway multisite LR entry vector with Pco-dCas9-VP64 and MS2-VP64 effectors for delivery to promoter sites for transcriptional activation. 5′ FLAG immuno-epitope tag, 5′ and 3′ nuclear localization signal (NLS), translational skipping mechanism T2A peptide, MS2 RNA-binding protein fused to NLS, and activator effector VP64. attL1 and attR5 sites flank the construct for Gateway LR cloning. (B) pYPQ143 Gateway multisite LR entry vector designed for expression of multiplex gRNA 2.0 cassettes. The zoom-out below the gRNA cassettes shows a listing of different gRNA modules for expression of variable numbers of gRNAs under either U6 or U3 small RNA promoters for expression in dicots or monocots respectively. (C and D) Multiplex TALE activator (mTALE-Act) entry vectors. (C) pYPQ121 illustration of dual TALE expression entry vector describing TALE1 and TALE2 with VP64 C-terminal fusions and NLS and T2A elements. Note TALE1 uses FLAG immune epitope while TALE2 utilizes HA tag. attL1 and attR5 sites are used for Gateway entry LR reaction. (D) pYPQ127B is similar to pYPQ121 but TALE3 and TALE4 are expressed from this vector and attL5 and attL2 recombination sites are used to assemble this vector in a Gateway-compatible destination vector along with pYPQ121. Note the use of 2× 35S promoter for this vector. Molecular Plant 2018 11, 245-256DOI: (10.1016/j.molp.2017.11.010) Copyright © 2017 The Author Terms and Conditions

Figure 2 Comparison of Four CRISPR Systems for Transcriptional Activation in Arabidopsis. (A–C) CRISPR dCas9-VP64 assembled at the genomic target site using conventional gRNA architecture. qRT–PCR analysis of fold activation of mRNA for (B) PAP1 and (C) FIS2 showing gene transcriptional activation for architecture in (A). (D–F) PAP1 and FIS2 gene activation for CRISPR dCas9-VP64-EDLL using the conventional gRNA architecture. (G–I) PAP1 and FIS2 gene activation for CRISPR dCas9-VP64 and MS2-EDLL with the gRNA 2.0 architecture. (J–L). PAP1 and FIS2 gene activation for CRISPR dCas9-VP64 and MS2-VP64 with the gRNA 2.0 architecture. All data displayed are fold changes of mRNA as assayed by qRT–PCR and are relative to pooled control plants (-) shown in red. PAP1 data are shown in gold and FIS2 in blue. Error bars represent SDs of technical replicates (n = 3). Molecular Plant 2018 11, 245-256DOI: (10.1016/j.molp.2017.11.010) Copyright © 2017 The Author Terms and Conditions

Figure 3 Robust Transcriptional Activation by the CRISPR-Act2.0 System in Rice. (A) qRT–PCR analysis of fold activation for Os03g01240 in rice protoplasts for comparison of dCas9-VP64 and CRISPR-Act2.0 systems; two gRNAs were multiplexed for targeting the promoter of Os03g01240. (B) qRT–PCR analysis of fold activation for Os04g39780 in rice protoplasts for comparison of dCas9-VP64 and CRISPR-Act2.0 systems; two gRNAs were multiplexed for targeting the promoter of Os04g39780. (C) qRT–PCR analysis for simultaneous activation of three genes (Os03g01240, Os04g39780, and Os11g35410) in rice protoplasts for comparison of dCas9-VP64 and CRISPR-Act2.0 systems; three gRNAs were multiplexed, with each gRNA targeting one promoter of each gene. Error bars represent SDs of technical replicates (n = 3). Molecular Plant 2018 11, 245-256DOI: (10.1016/j.molp.2017.11.010) Copyright © 2017 The Author Terms and Conditions

Figure 4 Activation of PAP1 in Arabidopsis by Multiplexing Two TALE-VP64 Transcriptional Activators. (A) Dual TALE-VP64 transcriptional activators target promoter regions of PAP1. TALE-38 targets the sequence indicated and has a 5′ HA tag and 3′ VP64 fusion with NLS sequences flanking the TALE repeats domain. TALE-37 with similar fusions targets a different locus with sequence as indicated. (B) qRT–PCR analysis of fold activation taken from total RNA of three independent T1 transgenic mTALE-Act lines with vector #258 and empty vector control (-). Error bars represent SDs of technical replicates (n = 3). Molecular Plant 2018 11, 245-256DOI: (10.1016/j.molp.2017.11.010) Copyright © 2017 The Author Terms and Conditions

Figure 5 Simultaneous Targeting of Three Arabidopsis Genes with TALE-VP64 Transcriptional Activators in T2 Lines. (A) mTALE-Act triplex of TALE activators delivered by T-DNA vector #94 with target locus sequence denoted below. Target genes are CSTF64, GL1, and RBP-DR1. The 5′ immuno-tags of HA or FLAG and 3′ fusions of VP64 effectors flank each TALE activator, as do NLS sequences. (B–D) qRT–PCR analysis of fold activation of mRNA for CSTF64, GL1, and RBP-DR1 in multiple T2 plants expressing TALE-VP64 illustrated in (A). (E) mTALE-Act triplex of TALE activators delivered by T-DNA vector #95, which targets three different sites. (F–H) qRT–PCR analysis of fold activation of mRNA for CSTF64, GL1, and RBP-DR1 in multiple T2 plants expressing TALE-VP64 illustrated in (E). Error bars represent SDs of technical replicates (n = 3). Molecular Plant 2018 11, 245-256DOI: (10.1016/j.molp.2017.11.010) Copyright © 2017 The Author Terms and Conditions

Figure 6 Probing Gene Regulation with Transcriptional Activation-Based Positive Feedback Loops. (A–D) Schematic of positive feedback transcriptional control loops. Native promoter activates dCas9-VP64-T2A-MS2-VP64 (A) or TALE-VP64 (B). The same activator will activate a gene of interest (GOI) or reporter under control of the same promoter. qRT–PCR analysis of fold change in transcriptional activation for miR319 using positive feedback circuit-based CRISPR-dCas9 (C) or TALE (D). (E) qRT–PCR analysis of mRNA fold change in gene activation for PAP1 using a positive feedback loop based on TALE. (F) Anthocyanin accumulation phenotype in Arabidopsis root tissue of highest activated PAP1 line (E; #260-6) and empty vector control seedlings. Zoom-in windows are labeled 1 or 2, indicating replicates. Purple roots are indicated by black arrows. Error bars represent SDs of technical replicates (n = 3). Molecular Plant 2018 11, 245-256DOI: (10.1016/j.molp.2017.11.010) Copyright © 2017 The Author Terms and Conditions