Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain.

DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF-TET1cd fusions, the SunTag-TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons

Co-targeting RNA Polymerases IV and V Promotes Efficient De Novo DNA Methylation in Arabidopsis.

The RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation

Symptom recovery in Tomato ringspot virus infected Nicotiana benthamiana plants : investigation into the role of plant RNA silencing mechanisms

Symptom recovery in virus-infected plants is characterized by the emergence of asymptomatic leaves after a systemic symptomatic phase of infection and has been linked with the clearance of the viral RNA due to the induction of RNA silencing. However, the recovery of Tomato ringspot virus (ToRSV)-infected Nicotiana benthamiana plants is not associated with viral RNA clearance in spite of active RNA silencing triggered against viral sequences. ToRSV isolate Rasp1-infected plants recover from infection at 27°C but not at 21°C, indicating a temperature-dependent recovery. In contrast, plants infected with ToRSV isolate GYV recover from infection at both temperatures. In this thesis, I studied the molecular mechanisms leading to symptom recovery in ToRSV-infected plants. I provide evidence that recovery of Rasp1-infected N. benthamiana plants at 27°C is associated with a reduction of the steady-state levels of RNA2-encoded coat protein (CP) but not of RNA2. In vivo labelling experiments revealed efficient synthesis of CP early in infection, but reduced RNA2 translation later in infection. Silencing of Argonaute1-like (NbAgo1) genes prevented both symptom recovery and RNA2 translation repression at 27°C. Also, translation repression was compromised in Rasp1-infected wild-type (WT) plants grown at 21°C. NbAgo1 and NbAgo2 mRNAs accumulated to similar levels at 21°C and 27°C in mock-inoculated WT plants. Both genes were induced during Rasp1 infection. Interestingly, the effect of silencing NbAgo2 on Rasp1 infection was only evident at low temperatures resulting in higher accumulation of CP. Taken together, our results suggest that although both NbAgo1 and NbAgo2 genes are induced, recovery of Rasp1-infected plants at 27°C is associated with an NbAgo1-dependent mechanism that represses the translation of viral RNA2. In contrast, recovery of GYV-infected plants is associated with a reduction of viral RNA and CP levels at both temperatures. Moreover, silencing of either NbAgo1 or NbAgo2 did not prevent recovery of GYV-infected plants at 21°C. However, both GYV-infected NbAgo1 and NbAgo2-silenced plants accumulated higher level of CP in recovered leaves compared to control plants. In conclusion, this study identifies translation repression as a novel regulatory mechanism in recovery and suggests that different mechanisms may operate during recovery in an isolate and/or temperature-dependent manner.Science, Faculty ofBotany, Department ofGraduat

CRISPR-CAS mediated transcriptional control and epi-mutagenesis

Tools for sequence-specific DNA binding have opened the door to new approaches in investigating fundamental questions in biology and crop development. While there are several platforms to choose from, many of the recent advances in sequence-specific targeting tools are focused on developing Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR Associated (CRISPR-Cas)-based systems. Using a catalytically inactive Cas protein (dCas), this system can act as a vector for different modular catalytic domains (effector domains) to control a gene's expression or alter epigenetic marks such as DNA methylation. Recent trends in developing CRISPR-dCas systems include creating versions that can target multiple copies of effector domains to a single site, targeting epigenetic changes that, in some cases, can be inherited to the next generation in the absence of the targeting construct, and combining effector domains and targeting strategies to create synergies that increase the functionality or efficiency of the system. This review summarizes and compares DNA targeting technologies, the effector domains used to target transcriptional control and epi-mutagenesis, and the different CRISPR-dCas systems used in plants

CRISPR-based targeting of DNA methylation in Arabidopsis thaliana by a bacterial CG-specific DNA methyltransferase.

CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research

A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana.

Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing

Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain.

DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF-TET1cd fusions, the SunTag-TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons

Improving cassava bacterial blight resistance by editing the epigenome

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement