Expanding plant genome editing scope and profiles with CRISPR-FrCas9 systems targeting palindromic TA sites.

CRISPR-Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5'-NNTA-3' PAM, targeting more abundant palindromic TA sites in plant genomes than the 5'-NGG-3' PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5'-NNTA-3' PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR-Cas9 system. FrCas9 induces high-efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2-FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2-FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9-derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C-to-T and A-to-G base edits in rice plants. Whole-genome sequencing-based off-target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2-FrCas9 in plants, however, causes detectable guide RNA-independent off-target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR-FrCas9 system for targeted mutagenesis, large deletions, C-to-T base editing, and A-to-G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR-FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope.

Xmas-2 Protein, the Core Protein of the TREX-2 mRNA Export Complex, Does not Determine the Specificity of ras2 mRNA Binding by the Complex.

The TREX-2 complex of eukaryotes is responsible for the export of a wide range of mRNAs from the nucleus to the cytoplasm. Previously, we showed that a subunit of the D. melanogaster TREX-2 complex, the PCID2 protein, has a domain that specifically interacts with RNA. However, it remains unknown whether other components of the complex are involved in interaction with and recognition of the target mRNA. In the present study, we determined the role of Xmas-2, the core structural subunit of the complex, in the specific recognition of ras2 mRNA fragments. In this work, we showed that Xmas-2 interacts with ras2 mRNA independently of other subunits of the complex. We showed that RNA-binding domains are located in both the N-terminal domain and the C-terminal domain of Xmas-2. However, the interaction of the protein with ras2 mRNA fragments is independent of RNA sequence and structure and is nonspecific. Thus, the Xmas-2 subunit is not involved in the recognition of specific RNA sequences by the complex.

TREX-2-ORC Complex of D. melanogaster Participates in Nuclear Export of Histone mRNA.

The TREX-2-ORC protein complex of D. melanogaster is necessary for the export of the bulk of synthesized poly(A)-containing mRNA molecules from the nucleus to the cytoplasm through the nuclear pores. However, the role of this complex in the export of other types of RNA remains unknown. We have shown that TREX-2-ORC participates in the nuclear export of histone mRNAs: it associates with histone mRNPs, binds to histone H3 mRNA at the 3'-terminal part of the coding region, and participates in the export of histone mRNAs from the nucleus to the cytoplasm.

Point Mutations in the M Domain of PCID2 Impair Its Function in mRNA Export in Drosophila melanogaster.

The PCID2 protein is a component of the eukaryotic TREX-2 complex, which is responsible for mRNA export from the nucleus into the cytoplasm. We have previously shown that Drosophila melanogaster PCID2 is involved in specific mRNA recognition and identified the key amino acids responsible for its interaction with the ras2 RNA. In this work, point mutations of the amino acids were shown to disrupt the PCID2 interaction with cell RNAs and to distort the export of polyA-containing mRNAs from the nucleus into the cytoplasm in Drosophila cells.

PCID2 Subunit of the Drosophila TREX-2 Complex Has Two RNA-Binding Regions.

Drosophila PCID2 is a subunit of the TREX-2 mRNA nuclear export complex. Although the complex has long been studied in eukaryotes, it is still unclear how TREX-2 interacts with mRNA in multicellular organisms. Here, the interaction between Drosophila PCID2 and the ras2 RNA was studied by EMSA. We show that the C-terminal region of the WH domain of PCID2 specifically binds the 3'-noncoding region of the ras2 RNA. While the same region of PCID2 interacts with the Xmas-2 subunit of the TREX-2 complex, PCID2 interacts with RNA independently of Xmas-2. An additional RNA-binding region (M region) was identified in the N-terminal part of the PCI domain and found to bind RNA nonspecifically. Point mutations of evolutionarily conserved amino acid residues in this region completely abolish the PCID2-RNA interaction, while a deletion of the C-terminal domain only partly decreases it. Thus, the specific interaction of PCID2 with RNA requires nonspecific PCID2-RNA binding.

THSC/TREX-2 deficiency causes replication stress and genome instability in Caenorhabditis elegans.

Transcription is an essential process of DNA metabolism, yet it makes DNA more susceptible to DNA damage. THSC/TREX-2 is a conserved eukaryotic protein complex with a key role in mRNP biogenesis and maturation that prevents genome instability. One source of such instability is linked to transcription, as shown in yeast and human cells, but the underlying mechanism and whether this link is universal is still unclear. To obtain further insight into the putative role of the THSC/TREX-2 complex in genome integrity, we have used Caenorhabditis elegans mutants of the thp-1 and dss-1 components of THSC/TREX-2. These mutants show similar defective meiosis, DNA damage accumulation and activation of the DNA damage checkpoint. However, they differ from each other regarding replication defects, as determined by measuring dUTP incorporation in the germline. Interestingly, this specific thp-1 mutant phenotype can be partially rescued by overexpression of RNase H. Furthermore, both mutants show a mild increase in phosphorylation of histone H3 at Ser10 (H3S10P), a mark previously shown to be linked to DNA-RNA hybrid-mediated genome instability. These data support the view that both THSC/TREX-2 factors prevent transcription-associated DNA damage derived from DNA-RNA hybrid accumulation by separate means.

Interaction of mRNA with the C-Terminal Domain of PCID2, a Subunit of the TREX-2 Complex, Is Required for Its Export from the Nucleus to the Cytoplasm in Drosophila melanogaster.

Following the transcription step, the newly synthesized mRNA is exported from the nucleus to the cytoplasm and further to the translation site. The TREX-2 complex is involved in the step of mRNA export from the nucleus to the cytoplasm. This complex in Drosophila melanogaster consists of four proteins: Xmas-2, PCID2, ENY2, and Sem1p. In our work, we have shown that deletion of the C-terminal sequence of PCID2 leads to a decrease in the interaction of the protein with RNA and to impaired mRNA export from the nucleus to the cytoplasm in D. melanogaster.

The Xmas-2 Protein of Drosophila melanogaster Undergoes Cleavage into Two Fragments.

The TREX-2 complex integrates several stages of gene expression, such as transcriptional activation and mRNA export. In D. melanogaster, TREX-2 consists of four major proteins: Xmas-2, ENY2, PCID2, and Sem1p. The Xmas-2 protein is the core subunit of the complex, with which other TREX-2 subunits interact. Xmas-2 homologues were found in all higher eukaryotes. Previously, it was shown that the human Xmas-2 homologue, GANP protein, can undergo cleavage into two parts, probably during apoptosis. We showed that the Xmas-2 protein of D. melanogaster can also split into two fragments. The resulting fragments of the protein correspond to the two large Xmas-2 domains. Protein splitting is observed both in vivo and in vitro. However, Xmas-2 cleavage in D. melanogaster is observed under normal conditions and is probably a part of the mechanism of transcription and mRNA export regulation in D. melanogaster.

The Human TREX-2 Complex Interacts with Subunits of the ORC Complex.

The TREX-2 protein complex is the key complex involved in the export of mRNA from the nucleus to the cytoplasm through the nuclear pores. Previously, a joint protein complex of TREX-2 with ORC was isolated in D. melanogaster. It was shown that the interaction of TREX-2 with ORC is necessary for efficient mRNA export from the nucleus to the cytoplasm. In this work, we showed that the TREX-2-ORC joint complex is also formed in human cells.

Nuclear mRNA Export and Aging.

The relationship between transcription and aging is one that has been studied intensively and experimentally with diverse attempts. However, the impact of the nuclear mRNA export on the aging process following its transcription is still poorly understood, although the nuclear events after transcription are coupled closely with the transcription pathway because the essential factors required for mRNA transport, namely TREX, TREX-2, and nuclear pore complex (NPC), physically and functionally interact with various transcription factors, including the activator/repressor and pre-mRNA processing factors. Dysregulation of the mediating factors for mRNA export from the nucleus generally leads to the aberrant accumulation of nuclear mRNA and further impairment in the vegetative growth and normal lifespan and the pathogenesis of neurodegenerative diseases. The optimal stoichiometry and density of NPC are destroyed during the process of cellular aging, and their damage triggers a defect of function in the nuclear permeability barrier. This review describes recent findings regarding the role of the nuclear mRNA export in cellular aging and age-related neurodegenerative disorders.

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis.

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.

PCID2, a subunit of the Drosophila TREX-2 nuclear export complex, is essential for both mRNA nuclear export and its subsequent cytoplasmic trafficking.

The TREX-2 complex is essential for the general nuclear mRNA export in eukaryotes. TREX-2 interacts with the nuclear pore and transcriptional apparatus and links transcription to the mRNA export. However, it remains poorly understood how the TREX-2-dependent nuclear export is connected to the subsequent stages of mRNA trafficking. Here, we show that the PCID2 subunit of Drosophila TREX-2 is present in the cytoplasm of the cell. The cytoplasmic PCID2 directly interacts with the NudC protein and this interaction maintains its stability in the cytoplasm. Moreover, PCID2 is associated with the cytoplasmic mRNA and microtubules. The PCID2 knockdown blocks nuclear export of mRNA and also affects the general mRNA transport into the cytoplasm. These data suggest that PCID2 could be the link between the nuclear TREX-2-dependent export and the subsequent cytoplasmic trafficking of mRNA.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana.

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp-CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein-peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Functional Validation of cas9/guideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8'OH1 loci.

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.

Study of the Interaction between Xmas-2, the Main Protein of TREX-2 mRNA Export Complex, and the Orc3 Protein, a Subunit of ORC Complex of D. melanogaster.

The TREX-2 protein complex is the key participant in the export of mRNA from the nucleus to the cytoplasm through the nuclear pores. Previously, a protein complex of D. melanogaster consisting of TREX-2 and ORC complexes was purified. It was shown that, in the TREX-2-ORC complex, the Xmas-2 protein, which is the platform for TREX-2 assembly, interacts with the Orc3 protein. The aim of this work was to investigate what regions of the Xmas-2 amino acid sequence are involved in the interaction with Orc3. It was shown that the interaction of  Xmas-2 with Orc3 requires a C-terminal region of  Xmas-2 located downstream of the CID domain.

First-generation genome editing in potato using hairy root transformation.

Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.

Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation.

Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 µm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 µm) as compared to those from the control group (178.9 ± 9.0 µm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.

Structure and Function of the TREX-2 Complex.

The Three prime repair exonuclease 2 (TREX-2) complex functions as a platform to which many of the components of the nuclear mRNA processing machinery bind, facilitating integration of this phase of the gene expression pathway, as well as mediating the re-positioning of highly regulated actively transcribing genes (such as GAL1) to nuclear pores (NPCs) to accelerate their activation. In Saccharomyces cerevisiae the TREX-2 complex is based on a Sac3 scaffold to which Thp1, Sem1, Cdc31 and two Sus1 chains are bound. A combination of X-ray crystallography and electron microscopy studies have established the structure of two major regions of this complex: the M-region that functions to bind nucleic acids and the CID region that functions to link the complex to nuclear pores. These structures have facilitated the engineering of mutants that have been used to define the contributions made by the TREX-2 complex to locating high-expressed genes to nuclear pores and the contributions made to mRNA nuclear export.

The Xmas-2 Homologues, the Main Component of the TREX-2 mRNA Export Complex.

TREX-2 complex is responsible for general mRNA export from nucleus to cytoplasm in eukaryote. The main protein of TREX-2 complex of D. melanogaster is protein Xmas-2. Its homologues in yeast and humans are Sac3 and GANP proteins, respectively. All three proteins contain the highly conserved domain Sac3-GANP, which is essential for interaction of TREX-2 complex with mRNA and another protein of the complex, PCID2. We identified two Xmas-2 homologues in D. melanogaster using the Sac3-GANP family domain sequence. These proteins have a common domain responsible for interaction with the PCID2 protein and RNA and are present in other eukaryotes. The function of these proteins is unknown. However, on the basis of their structural organization, we can assume that they interact with nucleic acids.

Nucleocytosolic mRNA transport in plants: export factors and their influence on growth and development.

In eukaryotes, the regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step in the expression of protein-coding genes, as it links nuclear mRNA synthesis with cytosolic translation. The pre-mRNAs that are synthesised by RNA polymerase II are processed by 5´-capping, splicing, and 3´-polyadenylation. The multi-subunit THO/TREX complex integrates mRNA biogenesis with their nucleocytosolic transport. Various export factors are recruited to the mRNAs during their maturation, which occurs essentially co-transcriptionally. These RNA-bound export factors ensure efficient transport of the export-competent mRNAs through nuclear pore complexes. In recent years, several factors involved in plant mRNA export have been functionally characterised. Analysis of mutant plants has demonstrated that impaired mRNA export causes defects in growth and development. Moreover, there is accumulating evidence that mRNA export can influence processes such as plant immunity, circadian regulation, and stress responses. Therefore, it is important to learn more details about the mechanism of nucleocytosolic mRNA transport in plants and its physiological significance.