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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co-expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA-gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen-activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR-edited mutants. The true knock-out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR-induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45-86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene-free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic variants in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNAs) need to be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and increased insertion size lower the efficiency to make a construct. Here, we report a strategy to quickly assembly multiple sgRNAs in one construct using a polycistronic-tRNA-gRNA (PTG) strategy. Taking advantage of the endogenous tRNA processing system in mammalian cells, we efficiently express multiple sgRNAs driven using only one Pol III promoter. Using an all-in-one construct carrying PTG, we disrupt the deacetylase domain in multiple histone deacetylases (HDACs) in human cells simultaneously. We demonstrate that multiple HDAC deletions significantly affect the activation of the Wnt- signaling pathway. Thus, this method enables to efficiently target multiple genes and provide a useful tool to establish mutated cells mimicking human diseases. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery. [ABSTRACT FROM AUTHOR]

Six NAC transcription factor genes were found to be targeted by miR164 at a highly conserved region for transactivation activity and to regulate drought tolerance negatively at the reproductive stage in rice.MicroRNAs constitute a large group of endogenous small RNAs of ~22 nt that emerge as vital regulators, mainly by targeting mRNAs for post-transcriptional repression. Previous studies have revealed that the miR164 family in Arabidopsis is comprised of three members which guide the cleavage of the mRNAs of five NAC genes to modulate developmental processes. However, the functions of the miR164-targeted NAC genes in crops are poorly deciphered. In this study, the conserved features of six miR164-targeted NAC genes (OMTN1–OMTN6) in rice are described, and evidence is provided that four of them confer a negative regulatory role in drought resistance. OMTN proteins have the characteristics of typical NAC transcriptional factors. The miR164 recognition sites of the OMTN genes are highly conserved in rice germplasms. Deletion of the recognition sites impaired the transactivation activity, indicating that the conserved recognition sites play a crucial role in maintaining the function of the OMTN proteins. The OMTN genes were responsive to abiotic stresses, and showed diverse spatio-temporal expression patterns in rice. Overexpression of OMTN2, OMTN3, OMTN4, and OMTN6 in rice led to negative effects on drought resistance at the reproductive stage. The expression of numerous genes related to stress response, development, and metabolism was altered in OMTN2-, OMTN3-, OMTN4-, and OMTN6-overexpressing plants. Most of the up-regulated genes in the OMTN-overexpressing plants were down-regulated by drought stress. The results suggest that the conserved miR164-targeted NAC genes may be negative regulators of drought tolerance in rice, in addition to their reported roles in development. [ABSTRACT FROM AUTHOR]

Several letters to the editor are presented in response to articles in previous issues related to the RNA-guided genome editing (RGE) using the Streptococcus pyogenes, gRNA specificity in animals, reduction of Cas9 affinity at the target site by the Nucleotide mismatch.

Targeted gene mutation was successfully achieved in rice using the CRISPR–Cas9 system. Experimental analyses of mutation efficiency and off-target effect as well as genome-wide prediction of specific guide RNA seeds suggest that the CRISPR–Cas9 system is a simple and effective tool for plant functional genomics and crop improvement.Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR–Cas9 system. Three guide RNAs (gRNAs) with a 20–22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif (PAM). The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation (insertion or deletion) by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3–8%. In addition, the off-target effect of an engineered gRNA–Cas9 was found on an imperfectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA–Cas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR–Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants. [ABSTRACT FROM AUTHOR]

MicroRNAs are a class of endogenous small RNA molecules (20-24 nucleotides) that have pivotal roles in regulating gene expression mostly at posttranscriptional levels in plants. Plant microRNAs have been implicated in the regulation of diverse biological processes including growth and stress responses. However, the information about microRNAs in regulating abiotic stress responses in rice is limited. We optimized a one-tube stem-loop reverse transcription quantitative PCR (ST-RT qPCR) for high-throughput expression profiling analysis of microRNAs in rice under normal and stress conditions. The optimized ST-RT qPCR method was as accurate as small RNA gel blotting and was more convenient and time-saving than other methods in quantifying microRNAs. With this method, 41 rice microRNAs were quantified for their relative expression levels after drought, salt, cold, and abscisic acid (ABA) treatments. Thirty-two microRNAs showed induced or suppressed expression after stress or ABA treatment. Further analysis suggested that stress-responsive cis-elements were enriched in the promoters of stress-responsive microRNA genes. The expressions of five and seven microRNAs were significantly affected in the rice plant with defects in stress tolerance regulatory genes OsSKIPa and OsbZIP23, respectively. Some of the predicted target genes of these microRNAs were also related to abiotic stresses. We conclude that ST-RT qPCR is an efficient and reliable method for expression profiling of microRNAs and a significant portion of rice microRNAs participate in abiotic stress response and regulation. [ABSTRACT FROM AUTHOR]

Summary: Lipids and lipid metabolites have essential roles in plant–pathogen interactions. GDSL‐type lipases are involved in lipid metabolism modulating lipid homeostasis. Some plant GDSLs modulate lipid metabolism altering hormone signal transduction to regulate host‐defence immunity. Here, we functionally characterized a rice lipase, OsGELP77, promoting both immunity and yield. OsGELP77 expression was induced by pathogen infection and jasmonic acid (JA) treatment. Overexpression of OsGELP77 enhanced rice resistance to both bacterial and fungal pathogens, while loss‐of‐function of osgelp77 showed susceptibility. OsGELP77 localizes to endoplasmic reticulum and is a functional lipase hydrolysing universal lipid substrates. Lipidomics analyses demonstrate that OsGELP77 is crucial for lipid metabolism and lipid‐derived JA homeostasis. Genetic analyses confirm that OsGELP77‐modulated resistance depends on JA signal transduction. Moreover, population genetic analyses indicate that OsGELP77 expression level is positively correlated with rice resistance against pathogens. Three haplotypes were classified based on nucleotide polymorphisms in the OsGELP77 promoter where OsGELP77Hap3 is an elite haplotype. Three OsGELP77 haplotypes are differentially distributed in wild and cultivated rice, while OsGELP77Hap3 has been broadly pyramided for hybrid rice development. Furthermore, quantitative trait locus (QTL) mapping and resistance evaluation of the constructed near‐isogenic line validated OsGELP77, a QTL for broad‐spectrum disease resistance. In addition, OsGELP77‐modulated lipid metabolism promotes JA accumulation facilitating grain yield. Notably, the hub defence regulator OsWRKY45 acts upstream of OsGELP77 by initiating the JA‐dependent signalling to trigger immunity. Together, OsGELP77, a QTL contributing to immunity and yield, is a candidate for breeding broad‐spectrum resistant and high‐yielding rice. [ABSTRACT FROM AUTHOR]

Background: Two-tiered plant immune responses involve cross-talk among defense-responsive (DR) genes involved in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), effector-triggered immunity (ETI) and effector-triggered susceptibility (ETS). Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an important bacterial disease that causes serious threats to rice yield and quality. Transcriptomic profiling provides an effective approach for the comprehensive and large-scale detection of DR genes that participate in the interactions between rice and Xoc. Results: In this study, we used RNA-seq to analyze the differentially expressed genes (DEGs) in susceptible rice after inoculation with two naturally pathogenic Xoc strains, a hypervirulent strain, HGA4, and a relatively hypovirulent strain, RS105. First, bacterial growth curve and biomass quantification revealed that differential growth occurred beginning at 1 day post inoculation (dpi) and became more significant at 3 dpi. Additionally, we analyzed the DEGs at 12 h and 3 days post inoculation with two strains, representing the DR genes involved in the PTI and ETI/ETS responses, respectively. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on the common DEGs, which included 4380 upregulated and 4019 downregulated genes and 930 upregulated and 1383 downregulated genes identified for the two strains at 12 h post inoculation (hpi) and 3 dpi, respectively. Compared to those at 12 hpi, at 3 dpi the number of common DEGs decreased, while the degree of differential expression was intensified. In addition, more disease-related GO pathways were enriched, and more transcription activator-like effector (TALE) putative target genes were upregulated in plants inoculated with HGA4 than in those inoculated with RS105 at 3 dpi. Then, four DRs were randomly selected for the BLS resistance assay. We found that CDP3.10, LOC_Os11g03820, and OsDSR2 positively regulated rice resistance to Xoc, while OsSPX3 negatively regulated rice resistance. Conclusions: By using an enrichment method for RNA-seq, we identified a group of DEGs related to the two stages of response to the Xoc strain, which included four functionally identified DR genes. [ABSTRACT FROM AUTHOR]

Phospholipids are important components of biological membranes, participating in various biological processes, including plant development and responses to biotic and abiotic stresses. A previous study showed that mutation of the rice OsCDS5 (CDP‐DAG Synthase) gene alters lipid metabolism, causing enhanced abiotic stress responses, yellowing of leaves at the seedling stage and delayed plant development. Here, we observed that the Oscds5 mutant shows enhanced resistance to rice blast, bacterial blight and bacterial leaf streak. Mutation of OsCDS5 promotes production of reactive oxygen species and increases the expression level of multiple defence‐related genes. Transcriptomic analyses indicate that genes involved in responses to stress, biotic/abiotic stimuli and metabolic processes are highly upregulated and enriched in mutant Oscds5. Metabolomic analyses showed that differential metabolites were enriched in the lipid metabolic and tryptophan metabolic pathways. The decreased level of phosphatidylinositol and increased level of serotonin probably contribute to enhanced disease resistance of the Oscds5 mutant. Taken together, mutation of OsCDS5 enhances abiotic and biotic stress responses, and OsCDS5 may be a promising target for genetic engineering to enhance the resilience of rice to abiotic and biotic stresses simultaneously. [ABSTRACT FROM AUTHOR]

DNA test is broadly used in diagnosis of crop disease and identification of genetic modified organism (GMO) in agriculture. However, rapid, low-cost, user-friendly, and field-deployable DNA test method is still limit. Recently, the RNA programmable nuclease of CRISPR/Cas is engineered as a new nucleic acid detection platform, but their application in plant remains to explore. In this study, we evaluated the Cas12a-based DNA detection for crop disease diagnosis and GMO test. A total of 14 crRNAs were designed to target two Magnaporthe oryzae genes and a synthetic Cry1C gene which encodes Bacillus thuringiensis δ-endotoxin and has been used to develop transgenic rice cultivar (Bt-rice) in China. Using a fluorescent reporter, targeted genes were easily detected by LbCas12a after recombinase polymerase amplification (RPA) for all crRNAs, despite that the signal strength varied 2–3-folds between different crRNAs. We further combined the filter paper-based DNA extraction and lateral flow assay (LFA) with RPA-Cas12a for DNA detection. This optimized Cas12a diagnostics method is carried at body temperature and does not require extra instrument except filter paper and LFA strip. Our data show that rice blast pathogen and Bt-rice were efficiently identified from leaf disc samples using this optimized DNA test method with highly active crRNAs. Moreover, LbCas12a exhibited variable nuclease activities on different targets; therefore, highly active crRNA is critical for successful DNA test using Cas12a and LFA. Owing to its simplicity, efficiency, and low-cost, DNA test using CRISPR/Cas12a would be easily applied in field for crop disease diagnosis and GMO administration. [ABSTRACT FROM AUTHOR]

Key message: Modifications of multiple copies of the BnaSAD2 gene family with genomic editing technology result in higher stearic acid content in the seed of polyploidy rapeseed. Solid fats from vegetable oils are widely used in food processing industry. Accumulating data showed that stearic acid is more favorite as the major composite among the saturate fatty acids in solid fats in considerations of its effects on human health. Rapeseed is the third largest oil crop worldwide, and has potential to be manipulated to produce higher saturated fatty acids as raw materials of solid fats. Toward that end, we identified four SAD2 gene family members in B. napus genome and established spatiotemporal expression pattern of the BnaSAD2 members. Genomic editing technology was applied to mutate all the copies of BnaSAD2 in this allopolyploid species and mutants at multiple alleles were generated and characterized to understand the effect of each BnaSAD2 member on blocking desaturation of stearic acid. Mutations occurred at BnaSAD2.A3 resulted in more dramatic changes of fatty acid profile than ones on BnaSAD2.C3, BnaSAD2.A5 and BnaSAD2.C4. The content of stearic acid in mutant seeds with single locus increased dramatically with a range of 3.1–8.2%. Furthermore, combination of different mutated alleles of BnaSAD2 resulted in more dramatic changes in fatty acid profiles and the double mutant at BnaSAD2.A3 and BnaSAD2.C3 showed the most dramatic phenotypic changes compared with its single mutants and other double mutants, leading to 11.1% of stearic acid in the seeds. Our results demonstrated that the members of BnaSAD2 have differentiated in their efficacy as a Δ9-Stearoyl-ACP-Desaturase and provided valuable rapeseed germplasm for breeding high stearic rapeseed oil. [ABSTRACT FROM AUTHOR]

Summary: Plants have evolved complex signalling networks to regulate growth and defence responses under an ever‐changing environment. However, the molecular mechanisms underlying the growth‐defence tradeoff are largely unclear. We previously reported that rice CALCIUM‐DEPENDENT PROTEIN KINASE 18 (OsCPK18) and MITOGEN‐ACTIVATED PROTEIN KINASE 5 (OsMPK5) mutually phosphorylate each other and that OsCPK18 phosphorylates and positively regulates OsMPK5 to suppress rice immunity. In this study, we found that OsCPK18 and its paralog OsCPK4 positively regulate plant height and yield‐related traits. Further analysis reveals that OsCPK18 and OsMPK5 synergistically regulate defence‐related genes but differentially regulate development‐related genes. In vitro and in vivo kinase assays demonstrated that OsMPK5 phosphorylates C‐terminal threonine (T505) and serine (S512) residues of OsCPK18 and OsCPK4, respectively. The kinase activity of OsCPK18T505D, in which T505 was replaced by aspartic acid to mimic T505 phosphorylation, displayed less calcium sensitivity than that of wild‐type OsCPK18. Interestingly, editing the MAPK phosphorylation motif in OsCPK18 and its paralog OsCPK4, which deprives OsMPK5‐mediated phosphorylation but retains calcium‐dependent activation of kinase activity, simultaneously increases rice yields and immunity. This editing event also changed the last seven amino acid residues of OsCPK18 and attenuated its binding with OsMPK5. This study presents a new regulatory circuit that fine tunes the growth‐defence tradeoff by modulating OsCPK18/4 activity and suggests that CRISPR/Cas9‐mediated engineering phosphorylation pathways could simultaneously improve crop yield and immunity. [ABSTRACT FROM AUTHOR]

Summary: Histone acetylation is a critical epigenetic modification that regulates plant immunity. Fungal pathogens secrete effectors that modulate host immunity and facilitate infection, but whether fungal pathogens have evolved effectors that directly target plant histone acetylation remains unknown.Here, we identified a secreted protein, UvSec117, from the rice false smut fungus, Ustilaginoidea virens, as a key effector that can target the rice histone deacetylase OsHDA701 and negatively regulates rice broad‐spectrum resistance against rice pathogens. UvSec117 disrupts host immunity by recruiting OsHDA701 to the nucleus and enhancing OsHDA701‐modulated deacetylation, thereby reducing histone H3K9 acetylation levels in rice plants and interfering with defense gene activation.Host‐induced gene silencing of UvSec117 promotes rice resistance to U. virens, thus providing an alternative way for developing rice false smut‐resistant plants.This is the first direct evidence demonstrating that a fungal effector targets a histone deacetylase to suppress plant immunity. Our data provided insight into a counter‐defense mechanism in a plant pathogen that inactivates host defense responses at the epigenetic level. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR)−CRISPR-associated protein 9 (Cas9)-mediated gene editing is revolutionizing plant research and crop breeding. Here, we present an effective and streamlined pipeline for arrayed CRISPR library construction and demonstrate it is suitable for small- to large-scale genome editing in plants. This pipeline introduces artificial PCR fragment-length markers for distinguishing guide RNAs (gRNAs) (FLASH), and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time. The identities of gRNAs in Agrobacterium mixtures and transgenic plants can therefore be read out by detecting the FLASH tags, a process that requires only conventional PCR and gel electrophoresis rather than sequencing. We generated an arrayed CRISPR library targeting all 1,072 members of the receptor-like kinase (RLK) family in rice. One-shot transformation generated a mutant population that covers gRNAs targeting 955 RLK s, and 74.3% (710/955) of the target genes had three or more independent T 0 lines. Our results indicate that the FLASH tags act as bona fide surrogates for the gRNAs and are tightly (92.1%) associated with frameshift mutations in the target genes. In addition, the FLASH pipeline allows for rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes. Furthermore, we showed that the RLK mutant library enables rapid discovery of defense-related RLK genes. This study introduces an effective pipeline for arrayed CRISPR library construction and provides genome-wide rice RLK mutant resources for functional genomics. This study reports a fast and effective pipeline for small- to large-scale gene editing by using an arrayed CRISPR library in plants. As a proof of concept, a CRISPR library targeting 1,072 members of the receptor-like kinase (RLK) family in rice was generated, and the results showed that this CRISPR library enabled the rapid discovery of defense-related genes. [ABSTRACT FROM AUTHOR]

The discussion about CRISPR/Cas genome editing is focused mostly on technical aspects to improve productivity and climate resilience in major tree crops such as cocoa, coffee, and citrus. We suggest a solution to the largely ignored socioeconomic impacts for farmers, when new genome-edited varieties are introduced from the laboratory to the field. [ABSTRACT FROM AUTHOR]

Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics. [ABSTRACT FROM AUTHOR]

Key message: Genomic editing with CRISPR/Cas9 system can simultaneously modify multiple copies of theBnaFAD2 gene to develop novel variations in fatty acids profiles in polyploidy rapeseed. Fatty acid composition affects edible and processing quality of vegetable oil and has been one of the primary targets for genetic modification in oilseed crops including rapeseed (Brassica napus). Fatty acid desaturase 2 gene, FAD2, is a key player that affects three major fatty acids, namely oleic, linoleic and linolenic acid, in oilseed plants. Previously, we showed that there are four copies of BnaFAD2 in allotetraploid rapeseed. In this study, we further established spatiotemporal expression pattern of each copy of BnaFAD2 using published RNA-seq data. Genomic editing technology based on CRISPR/Cas9 system was used to mutate all the copies of BnaFAD2 to create novel allelic variations in oleic acid and other fatty acid levels. A number of mutants at two targeting sites were identified, and the phenotypic variation in the mutants was systematically evaluated. The oleic acid content in the seed of the mutants increased significantly with the highest exceeding 80% compared with wild type of 66.43%, while linoleic and linolenic acid contents decreased accordingly. Mutations on BnaFAD2.A5 caused more dramatic changes of fatty acid profile than the mutations on BnaFAD2.C5 alleles that were identified with gene editing technique for the first time. Moreover, combining different mutated alleles of BnaFAD2 can even broaden the variation more dramatically. It was found that effects of different mutation types at BnaFAD2 alleles on oleic levels varied, indicating a possibility to manipulate fatty acid levels by precise mutation at specific region of a gene. [ABSTRACT FROM AUTHOR]

Summary: Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR‐Cas system, has led to significant progress in the field of plant and animal breeding. The CRISPR‐Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR‐Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre‐clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR‐Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR‐Cas‐modified organisms/products. [ABSTRACT FROM AUTHOR]

Summary: Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non‐vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co‐delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families. Significance Statement: In Physcomitrella patens, functional analysis, especially the analysis of genes from different gene families or genes with redundant functions in the same gene family, is hampered to some extent by the complexity and redundancy of these genes. The multiplex gene editing method reported here facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families, without the need for a laborious hybridization step. [ABSTRACT FROM AUTHOR]

Summary: Multilocular silique is a desirable agricultural trait with great potential for the development of high‐yield varieties of Brassica. To date, no spontaneous or induced multilocular mutants have been reported in Brassica napus, which likely reflects its allotetraploid nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed homologues of CLAVATA3 (CLV3) for a secreted peptide and its related receptors CLV1 and CLV2 in the CLV signalling pathway using the CRISPR/Cas9 system and achieved stable transmission of the mutations across three generations. Each BnCLV gene has two copies located in two subgenomes. The multilocular phenotype can be recovered only in knockout mutations of both copies of each BnCLV gene, illustrating that the simultaneous alteration of multiple gene copies by CRISPR/Cas9 mutagenesis has great potential in generating agronomically important mutations in rapeseed. The mutagenesis efficiency varied widely from 0% to 48.65% in T0 with different single‐guide RNAs (sgRNAs), indicating that the appropriate selection of the sgRNA is important for effectively generating indels in rapeseed. The double mutation of BnCLV3 produced more leaves and multilocular siliques with a significantly higher number of seeds per silique and a higher seed weight than the wild‐type and single mutant plants, potentially contributing to increased seed production. We also assessed the efficiency of the horizontal transfer of Cas9/gRNA cassettes by pollination. Our findings reveal the potential for plant breeding strategies to improve yield traits in currently cultivated rapeseed varieties. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeat (CRISPR) system has emerged as the revolutionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector (e.g., Cas9 and Cpf1) to a DNA target. Here, we elaborate a general strategy to simultaneously express multiple guide RNAs (gRNA) and CRISPR RNAs (crRNA) from introns of Cas9 and Cpf1. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpf1 to cleave the spliced intron that contains tRNA-gRNA polycistron or crRNA-crRNA array. We demonstrated that the tRNA-gRNA intron is able to fuse with Cas9 as one gene. Such a hybrid gene could be expressed using one polymerase II promoter, and exhibited high efficiency and robustness in simultaneously targeting multiple sites. We also implemented this strategy in Cpf1-mediated genome editing using intronic tRNA-crRNA and crRNA-crRNA arrays. Interestingly, hybrid genes containing Cpf1 and intronic crRNA array exhibited remarkably increased efficiency compared with the conventional Cpf1 vectors. Taken together, this study presents a method to express CRISPR reagents from one hybrid gene to increase genome-editing efficiency and capacity. Owing to its simplicity and versatility, this method could be broadly used to develop sophisticated CRISPR tools in eukaryotes. [ABSTRACT FROM AUTHOR]

Genome editing technology has rapidly evolved to knock-out genes, create targeted genetic variation, install precise insertion/deletion and single nucleotide changes, and perform large-scale alteration. The flexible and multipurpose editing technologies have started playing a substantial role in the field of plant disease management. CRISPR-Cas has reduced many limitations of earlier technologies and emerged as a versatile toolbox for genome manipulation. This review summarizes the phenomenal progress of the use of the CRISPR toolkit in the field of plant pathology. CRISPR-Cas toolbox aids in the basic studies on host-pathogen interaction, in identifying virulence genes in pathogens, deciphering resistance and susceptibility factors in host plants, and engineering host genome for developing resistance. We extensively reviewed the successful genome editing applications for host plant resistance against a wide range of biotic factors, including viruses, fungi, oomycetes, bacteria, nematodes, insect pests, and parasitic plants. Recent use of CRISPR-Cas gene drive to suppress the population of pathogens and pests has also been discussed. Furthermore, we highlight exciting new uses of the CRISPR-Cas system as diagnostic tools, which rapidly detect pathogenic microorganism. This comprehensive yet concise review discusses innumerable strategies to reduce the burden of crop protection. • Array of CRISPR tools are available for correcting pathogenic mutations in plant genomes. • CRISPR-mediated functional genomics identify virulence genes in pathogens as well as susceptibility and resistant genes in plants. • CRISPR-toolkit has been successfully applied for developing resistance against myriads of plant pathogens and pests. • CRISPR-based diagnostic platforms are rapid, accurate, and field-deployable for the early detection of plant diseases. • CRISPR tools have tremendous potential in engineering plant genomes for developing novel disease management strategies. [ABSTRACT FROM AUTHOR]

Theindicasubspecies of cultivated rice occupies the largest area of rice production in the world. However, a systematic analysis of cDNA sequences from theindicasubspecies has not been performed. The aim of the present study was to collect and analyze the expressed sequence tags (ESTs) ofindicarice on a large scale. A total of 39 208 raw sequences were generated from a normalized cDNA library prepared by use of 15 different tissues of theindicacultivar Minghui 63. After trimming, processing and analysis, 17 835 unique sequences were obtained, each of which presumably represents a unique gene. Of these sequences, 2663 were novel, and at least 70 wereindicaspecific. Comparison of the Minghui 63 sequences with the ESTs/full-length cDNAs in GenBank revealed a large number of deletion/insertion/substitution (DIS) at both the inter- and intra-subspecific levels. The overall number of polymorphisms in the expressed sequences was higher in the inter-subspecific comparisons than in the intra-subspecific comparisons. However, the extent of DIS-based polymorphism was highly variable among different rice varieties. In total, 15 726 unique sequences, including 697 novel sequences, were assigned to regions where large numbers of quantitative trait loci (QTLs) for agronomic traits had been detected previously. These results may be useful for developing new molecular markers for genetic mapping, detecting allelic polymorphisms associated with phenotypic variations between rice varieties, and facilitating QTL cloning by providing the starting points for candidate-gene identification. [ABSTRACT FROM AUTHOR]