Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration.

Insertion of a specific sequence in a targeted region for precise editing is still a major challenge in plants. Current protocols rely on inefficient homology-directed repair or non-homologous end-joining with modified double-stranded oligodeoxyribonucleotides (dsODNs) as donors. We developed a simple protocol that eliminates the need for expensive equipment, chemicals, modifications of donor DNA, and complicated vector construction. The protocol uses polyethylene glycol (PEG)-calcium to deliver low-cost, unmodified single-stranded oligodeoxyribonucleotides (ssODNs) and CRISPR/Cas9 ribonucleoprotein (RNP) complexes into Nicotiana benthamiana protoplasts. Regenerated plants were obtained from edited protoplasts with an editing frequency of up to 50% at the target locus. The inserted sequence was inherited to the next generation; this method thus opens the possibility for the future exploration of genomes by targeted insertion in plants.

Application of Protoplast Regeneration to CRISPR/Cas9 Mutagenesis in Nicotiana tabacum.

Protoplast transfection is widely used in plant research to rapidly evaluate RNA degradation, reporter assay, gene expression, subcellular localization, and protein-protein interactions. In order to successfully use protoplast transfection with the newly emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein editing platform, high yield of protoplasts, stable transfection efficiency, and reliable regeneration protocols are necessary. The Nicotiana tabacum transient protoplast transfection and regeneration system can effectively obtain target gene mutations in regenerated plants without transgenes and is thus a very attractive technique for evaluating gene editing reagents using CRISPR/Cas-based systems. Here, we describe in detail sterilized seed germination, culture conditions, isolation of Nicotiana tabacum protoplasts from tissue culture explants, construction of a vector containing the Cas protein and sgRNA cassette, highly efficient polyethylene glycol-calcium transient transfection of plasmids delivered into protoplasts, evaluation of mutagenesis efficiency and genotype analysis from protoplasts and regenerated plants, and the regeneration conditions to obtain CRISPR-edited plants from single protoplasts.

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration.

Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving CRISPR and Cas proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification, or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60 nt donor containing 27 nt in each homologous arm, 6/22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6 bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.

Genome Editing and Protoplast Regeneration to Study Plant-Pathogen Interactions in the Model Plant Nicotiana benthamiana.

Biotic diseases cause substantial agricultural losses annually, spurring research into plant pathogens and strategies to mitigate them. Nicotiana benthamiana is a commonly used model plant for studying plant-pathogen interactions because it is host to numerous plant pathogens and because many research tools are available for this species. The clustered regularly interspaced short palindromic repeats (CRISPR) system is one of several powerful tools available for targeted gene editing, a crucial strategy for analyzing gene function. Here, we demonstrate the use of various CRISPR-associated (Cas) proteins for gene editing of N. benthamiana protoplasts, including Staphylococcus aureus Cas9 (SaCas9), Streptococcus pyogenes Cas9 (SpCas9), Francisella novicida Cas12a (FnCas12a), and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID). We successfully mutated Phytoene Desaturase (PDS) and Ethylene Receptor 1 (ETR1) and the disease-associated genes RNA-Dependent RNA Polymerase 6 (RDR6), and Suppressor of Gene Silencing 3 (SGS3), and confirmed that the mutated alleles were transmitted to progeny. sgs3 mutants showed the expected phenotype, including absence of trans-acting siRNA3 (TAS3) siRNA and abundant expression of the GFP reporter. Progeny of both sgs3 and rdr6 null mutants were sterile. Our analysis of the phenotypes of the regenerated progeny indicated that except for the predicted phenotypes, they grew normally, with no unexpected traits. These results confirmed the utility of gene editing followed by protoplast regeneration in N. benthamiana. We also developed a method for in vitro flowering and seed production in N. benthamiana, allowing the regenerants to produce progeny in vitro without environmental constraints.

Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco.

KEY MESSAGE: Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.

Genome Sequences Provide Insights into the Reticulate Origin and Unique Traits of Woody Bamboos.

Polyploidization is a major driver of speciation and its importance to plant evolution has been well recognized. Bamboos comprise one diploid herbaceous and three polyploid woody lineages, and are members of the only major subfamily in grasses that diversified in forests, with the woody members having a tree-like lignified culm. In this study, we generated four draft genome assemblies of major bamboo lineages with three different ploidy levels (diploid, tetraploid, and hexaploid). We also constructed a high-density genetic linkage map for a hexaploid species of bamboo, and used a linkage-map-based strategy for genome assembly and identification of subgenomes in polyploids. Further phylogenomic analyses using a large dataset of syntenic genes with expected copies based on ploidy levels revealed that woody bamboos originated subsequent to the divergence of the herbaceous bamboo lineage, and experienced complex reticulate evolution through three independent allopolyploid events involving four extinct diploid ancestors. A shared but distinct subgenome was identified in all polyploid forms, and the progenitor of this subgenome could have been critical in ancient polyploidizations and the origin of woody bamboos. Important genetic clues to the unique flowering behavior and woody trait in bamboos were also found. Taken together, our study provides significant insights into ancient reticulate evolution at the subgenome level in the absence of extant donor species, and offers a potential model scenario for broad-scale study of angiosperm origination by allopolyploidization.

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration.

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

Global transcriptome analysis and identification of a CONSTANS-like gene family in the orchid Erycina pusilla.

The high chromosome numbers, polyploid genomes, and long juvenile phases of most ornamental orchid species render functional genomics difficult and limit the discovery of genes influencing horticultural traits. The orchid Erycina pusilla has a low chromosome number (2n = 12) and flowers in vitro within 1 year, making it a standout candidate for use as a model orchid. However, transcriptomic and genomic information from E. pusilla remains limited. In this study, next-generation sequencing (NGS) technology was used to identify 90,668 unigenes by de novo assembly. These unigenes were annotated functionally and analyzed with regard to their gene ontology (GO), clusters of orthologous groups (COG), and KEGG pathways. To validate the discovery methods, a homolog of CONSTANS (CO), one of the key genes in the flowering pathway, was further analyzed. The Arabidopsis CO-Like (COL) amino acid sequences were used to screen for homologs in the E. pusilla transcriptome database. Specific primers to the homologous unigenes were then used to isolate BAC clones, which were sequenced to identify 12 E. pusilla CO-like (EpCOL) full-length genes. Based on sequence homology, domain structure, and phylogenetic analysis, these EpCOL genes were divided into four groups. Four EpCOLs fused with GFP were localized in the nucleus. Some EpCOL genes were regulated by light. These results demonstrate that nascent E. pusilla resources (transcriptome and BAC library) can be used to investigate the E. pusilla photoperiod-dependent flowering genes. In future, this strategy can be applied to other biological processes, marketable traits, and molecular breeding in this model orchid.

Screening a cDNA library for protein-protein interactions directly in planta.

Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein- or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ~2 × 10(5) cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.

Anticancer effects of tanshinone I in human non-small cell lung cancer.

Tanshinones are the major bioactive compounds of Salvia miltiorrhiza Bunge (Danshen) roots, which are used in many therapeutic remedies in Chinese traditional medicine. We investigated the anticancer effects of tanshinones on the highly invasive human lung adenocarcinoma cell line, CL1-5. Tanshinone I significantly inhibited migration, invasion, and gelatinase activity in macrophage-conditioned medium-stimulated CL1-5 cells in vitro and also reduced the tumorigenesis and metastasis in CL1-5-bearing severe combined immunodeficient mice. Unlike tanshinone IIA, which induces cell apoptosis, tanshinone I did not have direct cytotoxicity. Real-time quantitative PCR, luciferase reporter assay, and electrophoretic mobility shift assay revealed that tanshinone I reduces the transcriptional activity of interleukin-8, the angiogenic factor involved in cancer metastasis, by attenuating the DNA-binding activity of activator protein-1 and nuclear factor-kappaB in conditioned medium-stimulated CL1-5 cells. Microarray and pathway analysis of tumor-related genes identified the differentially expressed genes responding to tanshinone I, which may be associated with the Ras-mitogen-activated protein kinase and Rac1 signaling pathways. These results suggest that tanshinone I exhibits anticancer effects both in vitro and in vivo and that these effects are mediated at least partly through the interleukin-8, Ras-mitogen-activated protein kinase, and Rac1 signaling pathways. Although tanshinone I has a remarkable anticancer action, its potential anticoagulant effect should be noted and evaluated.