CRISPR AND Plant

Understanding genomic functions requires site-specific manipulation of loci via efficient protein effector targeting systems. However, few approaches for targeted manipulation of the epigenome are available in plants. Here, we adapt the dCas9-SunTag system to engineer targeted gene activation and DNA methylation in Arabidopsis. We demonstrate that a dCas9-SunTag system utilizing the transcriptional activator VP64 drives robust and specific activation of several loci, including protein coding genes and transposable elements, in diverse chromatin contexts...

Although DNA-protein crosslinks (DPCs) represent a severe threat to the genome integrity, only recently main mechanisms of DPC repair were elucidated in humans and yeast. Here we define the pathways for DPC repair in plants. Using CRISPR/Cas9, we could show that only one of two homologues of the universal repair proteases SPRTN/WSS1 (WSS1A) is essential for DPC repair in Arabidopsis. WSS1A defective lines exhibit developmental defects and are hypersensitive to camptothecin (CPT) and cis-platin. Interestingly, the CRISPR/Cas9 mutants of the tyrosyl-DNA phosphodiesterase 1 (TDP1) are insensitive to CPT, and only the wss1A tdp1 double mutant reveals a higher sensitivity than the wss1A single mutant...

Precision crop breeding, using genome editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR) systems to improve useful traits in crop plants, holds great potential for the future of agriculture. Using CRISPR-Cas9, recent studies have engineered domestication traits in wild-relative species of tomato crop for higher nutritive value and better adaptation to diverse stresses.

Tomato (Solanum lycopersicum) is a model for climacteric fleshy fruit ripening studies. Tomato ripening is regulated by multiple transcription factors together with the plant hormone ethylene and their downstream effector genes. Transcription Factors APETALA2a (AP2a), NON-RIPENING (NOR) and FRUITFULL (FUL1/TDR4 and FUL2/MBP7) were reported as master regulators controlling tomato fruit ripening. Their proposed functions were derived from studies of the phenotype of spontaneous mutants or RNAi knock-down lines rather than, as it appears now, actual null mutants...

Malformin A1 (MA1) is a fungus-produced cyclic pentapeptide. MA1 exhibits teratogenicity to plants, fibrinolysis-enhancing activity, and cytotoxicity to mammalian cells. To clarify the cytotoxic mechanism of MA1, we screened for the genes involved in the cytotoxicity of MA1 in monocytoid U937 cells by using CRISPR/Cas9-based genome-wide knockout library. Screening was performed by positive selection for the cells resistant to MA1 treatment, and sgRNAs integrated into MA1-resistant cells were analyzed with high-throughput sequencing...

BACKGROUND: TRIANGULAR HULL1 (TH1), a member of the rice ALOG gene family, has been characterized as a rice lemma/palea-related gene. To understand the gene regulatory network that controlled by TH1, we analyzed the transcriptome from a TH1 knock out (KO) line, which was generated by CRISPR/Cas9. Our study may shed some light on the molecular mechanism of lemma/palea development. RESULTS: We obtained 20 T0 th1-C transgenic plants by CRISPR/Cas9. Among the 20 plants, there were eight bi-allelic mutations, five homozygous mutations, three heterozygous mutations, and four Non-KO plants...

The arrival of sequence-specific endonucleases that allow genome editing has shaken the pillars of basic and applied plant biology. Clustered regularly interspaced palindromic repeats (CRISPR) is a revolutionary genome-engineering tool that enables the enhancement of targeted traits in plants. Numerous plants, including energy crops, known for their potential to tolerate, immobilize, and stabilize inorganic and organic pollutants, have already been edited using different CRISPR systems. Moreover, a large array of genes responsible for increased metal tolerance, metal uptake and hyperaccumulation have already been identified...

Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway...

New Plant Breeding Technologies (NPBTs), including CRISPR gene editing, are widely used and drive the development of new crops. However, these new technologies are disputed, creating uncertainty in how applications of these technologies for agricultural and food uses will be regulated. While in North America regulatory systems respond with a differentiated approach to NPBTs, the Court of Justice of the European Union has in effect made most if not all NPBT subject to the same regulatory regime as GMOs. Since, discussion on the regulatory reform of the EU's GM legal systems are underway...

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with high copy transgenes and low edit rate in wheat, a large T0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects...

The rapid and widespread adoption of CRISPR/Cas technologies has allowed genetic editing in plants to enter a revolutionary new era. In this mini-review we highlight the current CRISPR/Cas tools available in plants and the use of Arabidopsis thaliana as a model to guide future improvements in crop yields, such as enhancing photosynthetic potential. We also outline the current socio-political landscape for CRISPR/Cas research and highlight the growing need for governments to better facilitate research into plant genetic editing technologies...

BACKGROUND: qPE9-1/DEP1, encoding a G protein γ subunit, has multiple effects on plant architecture, grain size, and yield in rice. The qPE9-1 protein contains an N-terminal G gamma-like (GGL) domain, a putative transmembrane domain, and a C-terminal cysteine-rich domain. However, the roles of each domain remain unclear. RESULTS: In the present study, we focused on the genetic effects of different domains of qPE9-1 in the regulation of grain length and weight. We generated a series of transgenic plants expressing different truncated qPE9-1 proteins through constitutive expression and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 strategies...

BACKGROUND: CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing. RESULTS: We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice...

MicroRNAs play important roles in posttranscriptional regulation of plant development, metabolism, and abiotic stress responses. The recent generation of massive amounts of small RNA sequence data, along with development of bioinformatic tools to identify miRNAs and their mRNA targets, has led to an explosion of newly identified putative miRNAs in plants. Genome editing techniques like CRISPR-Cas9 will allow us to study the biological role of these potential novel miRNAs by efficiently targeting both the miRNA and its mRNA target...

Viroid discovery as well as the economic significance of viroids and biological properties are presented. Next-generation sequencing (NGS) technologies combined with informatics have been applied to viroid research and diagnostics for almost a decade. NGS provides highly efficient, rapid, low-cost high-throughput sequencing of viroid genomes and of the 21⁻24 nt vd-sRNAs generated by the RNA silencing defense of the host. NGS has been utilized in various viroid studies which are presented. The discovery during the last few years that prokaryotes have heritable adaptive immunity mediated through clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated Cas proteins, have led to transformative advances in molecular biology, notably genome engineering and most recently molecular diagnostics...

Apomixis is an asexual reproduction way of plants that can produce clonal offspring through seeds. In this study, we introduced apomixis into rice (Oryza sativa) by mutating OsSPO11-1, OsREC8, OsOSD1, and OsMATL through a CRISPR/Cas9 system. The quadruple mutant showed a transformation from meiosis to mitosis and produced clonal diploid gametes. With mutated Osmatl, which gives rise to haploid induction in plants, the quadruple mutant is expected to be able to produced apomictic diploid offspring. We named this quadruple mutant as AOP (Apomictic Offspring Producer) for its ability to produce apomictic offspring...

Targeting-induced local lesions in genomes (TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 individuals...

BACKGROUND: Recent innovation in the field of genome engineering encompasses numerous levels of plant genome engineering which attract the substantial excitement of plant biologist worldwide. RNA-guided CRISPR Cas9 system has appeared a promising tool in site-directed mutagenesis due to its innovative utilization in different branches of biology. CRISPR-Cas9 nuclease system have supersedes all previously existed strategies and their associated pitfalls encountered with site-specific mutagenesis...

Grain size is a key determiner of grain weight, one of the yield components in rice (Oryza sativa). Therefore, to increase grain yield, it is important to elucidate the detailed mechanisms regulating grain size. The Large grain (Lgg) mutant, found in the nDart1-tagged lines of Koshihikari, is caused by a truncated nDart1-3 and 355 bp deletion in the 5' UTR of LGG, which encodes a putative RNA-binding protein, through transposon display and cosegregation analysis between grain length and LGG genotype in F2 and F3...

The analysis of 93 mutant alleles in 18 genes demonstrated that CRISPR-Cas9 is a robust tool for targeted mutagenesis in maize, permitting efficient generation of single and multiple knockouts. CRISPR-Cas9 technology is a simple and efficient tool for targeted mutagenesis of the genome. It has been implemented in many plant species, including crops such as maize. Here we report single- and multiple-gene mutagenesis via stably transformed maize plants. Two different CRISPR-Cas9 vectors were used allowing the expression of multiple guide RNAs and different strategies to knockout either independent or paralogous genes...