Editorial: CRISPR-Cas in Agriculture: Opportunities and Challenges

CRISPR-Cas genome editing technology is developing at a rapid pace and new molecular tools, such as CRISPR nucleases, are becoming regularly available. As part of this Research Topic, Bandyopadhyay et al. provide a comprehensive overview of Cas12a, a CRISPR nuclease formerly known as Cpf1. In their review article, the authors cover structural and mechanistic aspects of Cas12a in comparison with Cas9, the most commonly used CRISPR nuclease. They also highlight uses of Cas12a for the purpose of improving agriculturally important traits in various crops. An overview of Cas9 genome editing applications in plants is provided by El-Mounadi et al. who introduce the reader to the mechanism of Cas9 activity, methods of its delivery to plant cells (i.e., transformation techniques), give examples of improving crop traits using CRISPR-Cas9, and touch on biosafety and regulatory aspects associated with genome editing. A number of countries (e.g., the USA, Brazil, Argentina, and Japan) have already exempted genome edited crops, which do not carry transgenic DNA or novel combination of genetic material (i.e., not similarly achievable through conventional breeding), from being regulated similarly to Genetically Modified Organisms (GMOs) as genetically engineered (GE) organisms (Schmidt et al., 2020). Although the above-mentioned countries have passed legislation allowing cultivation of genome edited crops without GE regulation, the public dialogue and policy developments on the issue are evolving. In the case of Japan, Tabei et al. analyse Twitter conversation on genome-edited foods and their labelling over the period from May to October 2019. The analysis reveals that 54.5% of relevant tweets were statements opposed to food produced using genome edited crops, while only 7% were statements in favour of it. The remaining 38.5% of tweets were statements deemed neutral. Although the analysis was not necessarily representative of the wider Japanese society due to bias among Twitter users, the study underlines the importance of a continuous public dialogue on the issue of genome edited crops in Japan and the rest of the world

Adaptive evolution of chloroplast genome structure inferred using a parametric bootstrap approach

BACKGROUND: Genome rearrangements influence gene order and configuration of gene clusters in all genomes. Most land plant chloroplast DNAs (cpDNAs) share a highly conserved gene content and with notable exceptions, a largely co-linear gene order. Conserved gene orders may reflect a slow intrinsic rate of neutral chromosomal rearrangements, or selective constraint. It is unknown to what extent observed changes in gene order are random or adaptive. We investigate the influence of natural selection on gene order in association with increased rate of chromosomal rearrangement. We use a novel parametric bootstrap approach to test if directional selection is responsible for the clustering of functionally related genes observed in the highly rearranged chloroplast genome of the unicellular green alga Chlamydomonas reinhardtii, relative to ancestral chloroplast genomes. RESULTS: Ancestral gene orders were inferred and then subjected to simulated rearrangement events under the random breakage model with varying ratios of inversions and transpositions. We found that adjacent chloroplast genes in C. reinhardtii were located on the same strand much more frequently than in simulated genomes that were generated under a random rearrangement processes (increased sidedness; p < 0.0001). In addition, functionally related genes were found to be more clustered than those evolved under random rearrangements (p < 0.0001). We report evidence of co-transcription of neighboring genes, which may be responsible for the observed gene clusters in C. reinhardtii cpDNA. CONCLUSION: Simulations and experimental evidence suggest that both selective maintenance and directional selection for gene clusters are determinants of chloroplast gene order

The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

Noncoding RNA Mediated Traffic of Foreign mRNA into Chloroplasts Reveals a Novel Signaling Mechanism in Plants

Communication between chloroplasts and the nucleus is one of the milestones of the evolution of plants on earth. Proteins encoded by ancestral chloroplast-endogenous genes were transferred to the nucleus during the endosymbiotic evolution and originated this communication, which is mainly dependent on specific transit-peptides. However, the identification of nuclear-encoded proteins targeted to the chloroplast lacking these canonical signals suggests the existence of an alternative cellular pathway tuning this metabolic crosstalk. Non-coding RNAS (NcRNAs) are increasingly recognized as regulators of gene expression as they play roles previously believed to correspond to proteins. Avsunviroidae family viroids are the only noncoding functional RNAs that have been reported to traffic inside the chloroplasts. Elucidating mechanisms used by these pathogens to enter this organelle will unearth novel transport pathways in plant cells. Here we show that a viroid-derived NcRNA acting as a 5′UTR-end mediates the functional import of Green Fluorescent Protein (GFP) mRNA into chloroplast. This claim is supported by the observation at confocal microscopy of a selective accumulation of GFP in the chloroplast of the leaves expressing the chimeric vd-5′UTR/GFP and by the detection of the GFP mRNA in chloroplasts isolated from cells expressing this construct. These results support the existence of an alternative signaling mechanism in plants between the host cell and chloroplasts, where an ncRNA functions as a key regulatory molecule to control the accumulation of nuclear-encoded proteins in this organelle. In addition, our findings provide a conceptual framework to develop new biotechnological tools in systems using plant chloroplast as bioreactors. Finally, viroids of the family Avsunviroidae have probably evolved to subvert this signaling mechanism to regulate their differential traffic into the chloroplast of infected cells

Screening non-coding RNAs in transcriptomes from neglected species using PORTRAIT: case study of the pathogenic fungus Paracoccidioides brasiliensis

<p>Abstract</p> <p>Background</p> <p>Transcriptome sequences provide a complement to structural genomic information and provide snapshots of an organism's transcriptional profile. Such sequences also represent an alternative method for characterizing neglected species that are not expected to undergo whole-genome sequencing. One difficulty for transcriptome sequencing of these organisms is the low quality of reads and incomplete coverage of transcripts, both of which compromise further bioinformatics analyses. Another complicating factor is the lack of known protein homologs, which frustrates searches against established protein databases. This lack of homologs may be caused by divergence from well-characterized and over-represented model organisms. Another explanation is that non-coding RNAs (ncRNAs) may be caught during sequencing. NcRNAs are RNA sequences that, unlike messenger RNAs, do not code for protein products and instead perform unique functions by folding into higher order structural conformations. There is ncRNA screening software available that is specific for transcriptome sequences, but their analyses are optimized for those transcriptomes that are well represented in protein databases, and also assume that input ESTs are full-length and high quality.</p> <p>Results</p> <p>We propose an algorithm called PORTRAIT, which is suitable for ncRNA analysis of transcriptomes from poorly characterized species. Sequences are translated by software that is resistant to sequencing errors, and the predicted putative proteins, along with their source transcripts, are evaluated for coding potential by a support vector machine (SVM). Either of two SVM models may be employed: if a putative protein is found, a protein-dependent SVM model is used; if it is not found, a protein-independent SVM model is used instead. Only <it>ab initio </it>features are extracted, so that no homology information is needed. We illustrate the use of PORTRAIT by predicting ncRNAs from the transcriptome of the pathogenic fungus <it>Paracoccidoides brasiliensis </it>and five other related fungi.</p> <p>Conclusion</p> <p>PORTRAIT can be integrated into pipelines, and provides a low computational cost solution for ncRNA detection in transcriptome sequencing projects.</p

High-Throughput Sequencing, Characterization and Detection of New and Conserved Cucumber miRNAs

Micro RNAS (miRNAs) are a class of endogenous small non coding RNAs involved in the post-transcriptional regulation of gene expression. In plants, a great number of conserved and specific miRNAs, mainly arising from model species, have been identified to date. However less is known about the diversity of these regulatory RNAs in vegetal species with agricultural and/or horticultural importance

RNA degradome—its biogenesis and functions

RNA degradation is among the most fundamental processes that occur in living cells. The continuous decay of RNA molecules is associated not only with nucleotide turnover, but also with transcript maturation and quality control. The efficiency of RNA decay is ensured by a broad spectrum of both specific and non-specific ribonucleases. Some of these ribonucleases participate mainly in processing primary transcripts and in RNA quality control. Others preferentially digest mature, functional RNAs to yield a variety of molecules that together constitute the RNA degradome. Recently, it has become increasingly clear that the composition of the cellular RNA degradome can be modulated by numerous endogenous and exogenous factors (e.g. by stress). In addition, instead of being hydrolyzed to single nucleotides, some intermediates of RNA degradation can accumulate and function as signalling molecules or participate in mechanisms that control gene expression. Thus, RNA degradation appears to be not only a process that contributes to the maintenance of cellular homeostasis but also an underestimated source of regulatory molecules

Widespread Polymorphism in the Positions of Stop Codons in Drosophila melanogaster

The mechanisms underlying evolutionary changes in protein length are poorly understood. Protein domains are lost and gained between species and must have arisen first as within-species polymorphisms. Here, we use Drosophila melanogaster population genomic data combined with between species divergence information to understand the evolutionary forces that generate and maintain polymorphisms causing changes in protein length in D. melanogaster. Specifically, we looked for protein length variations resulting from premature termination codons (PTCs) and stop codon losses (SCLs). We discovered that 438 genes contained polymorphisms resulting in truncation of the translated region (PTCs) and 119 genes contained polymorphisms predicted to lengthen the translated region (SCLs). Stop codon polymorphisms (SCPs) (especially PTCs) appear to be more deleterious than other polymorphisms, including protein amino acid changes. Genes harboring SCPs are in general less selectively constrained, more narrowly expressed, and enriched for dispensable biological functions. However, we also observed exceptional cases such as genes that have multiple independent SCPs, alleles that are shared between D. melanogaster and Drosophila simulans, and high-frequency alleles that cause extreme changes in gene length. SCPs likely have an important role in the evolution of these genes

Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon

BACKGROUND: The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon. RESULTS: B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. CONCLUSIONS: B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by BioMed Central Ltd. The published article can be found at: http://genomebiology.com/