RESUMEN
Extreme temperature conditions seriously impair male reproductive development in plants; however, the molecular mechanisms underlying the response of anthers to extreme temperatures remain poorly described. The transcription factor phytochrome-interacting factor4 (PIF4) acts as a hub that integrates multiple signaling pathways to regulate thermosensory growth and architectural adaptation in plants. Here, we report that SlPIF4 in tomato (Solanum lycopersicum) plays a pivotal role in regulating cold tolerance in anthers. CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9-generated SlPIF4 knockout mutants showed enhanced cold tolerance in pollen due to reduced temperature sensitivity of the tapetum, while overexpressing SlPIF4 conferred pollen abortion by delaying tapetal programmed cell death (PCD). SlPIF4 directly interacts with SlDYT1, a direct upstream regulator of SlTDF1, both of which (SlDYT1 and SlTDF1) play important roles in regulating tapetum development and tapetal PCD. Moderately low temperature (MLT) promotes the transcriptional activation of SlTDF1 by the SlPIF4-SlDYT1 complex, resulting in pollen abortion, while knocking out SlPIF4 blocked the MLT-induced activation of SlTDF1. Furthermore, SlPIF4 directly binds to the canonical E-box sequence in the SlDYT1 promoter. Collectively, these findings suggest that SlPIF4 negatively regulates cold tolerance in anthers by directly interacting with the tapetal regulatory module in a temperature-dependent manner. Our results shed light on the molecular mechanisms underlying the adaptation of anthers to low temperatures.
Asunto(s)
Solanum lycopersicum/metabolismo , Apoptosis/genética , Apoptosis/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , TemperaturaRESUMEN
Plant height is a key agronomic trait regulated by several phytohormones such as gibberellins (GAs) and auxin. However, little is known about how cytokinin (CK) participates in this process. Here, we report that SlRR6, a type-A response regulator in the CK signaling pathway, positively regulates plant height in tomato. SlRR6 was induced by exogenous kinetin and GA3, but inhibited by indole-3-acetic acid (IAA). Knock out of SlRR6 reduced tomato plant height through shortening internode length, while overexpression of SlRR6 caused taller plants due to increased internode number. Cytological observation of longitudinal stems showed that both knock out and overexpression of SlRR6 generated larger cells, but significantly reduced cell numbers in each internode. Further studies demonstrated that overexpression of SlRR6 enhanced GA accumulation and lowered IAA content, along with expression changes in GA- and IAA-related genes. Exogenous paclobutrazol and IAA treatments restored the increased plant height phenotype in SlRR6-overexpressing lines. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays showed that SlRR6 interacts with a small auxin up RNA protein, SlSAUR58. Moreover, SlSAUR58-overexpressing plants were dwarf with decreased internode length. Overall, our findings establish SlRR6 as a vital component in the CK signaling, GA, and IAA regulatory network that controls plant height.
Asunto(s)
Giberelinas , Solanum lycopersicum , Giberelinas/metabolismo , Citocininas/metabolismo , Solanum lycopersicum/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Ácidos Indolacéticos/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
The development of viable pollen determines male fertility, and is crucial for reproduction in flowering plants. Phytochrome interacting factor 3 (PIF3) acts as a central regulator of plant growth and development, but its relationship with pollen development has not been determined. Through genetic, histological and transcriptomic analyses, we identified an essential role for SlPIF3 in regulating tomato (Solanum lycopersicum) pollen development. Knocking out SlPIF3 using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 resulted in pollen mitosis I arrest, and a failure to form viable pollen. We further demonstrated that both glutamate synthase 1 (SlGLT1) and cell wall invertase 9 (SlCWIN9), involved in auxin and sugar homeostasis, respectively, colocalised with SlPIF3 in the anthers and were directly regulated by SlPIF3. Knockout of either SlGLT1 or SlCWIN9 phenocopied the pollen phenotype of SlPIF3 knockout (Slpif3) lines. Slpif3 fertility was partially restored by exogenous auxin indole-3-acetic acid in a dose-dependent manner. This study reveals a mechanism by which SlPIF3 regulates pollen development and highlights a new strategy for creating hormone-regulated genic male sterile lines for tomato hybrid seed production.
Asunto(s)
Fitocromo , Solanum lycopersicum , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Solanum lycopersicum/metabolismo , Fitocromo/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/metabolismo , Azúcares/metabolismoRESUMEN
High temperature (HT) is becoming an increasingly serious factor in limiting crop production with global climate change. During hot seasons, owing to prevailing HT, cultivated tomatoes are prone to exhibiting stigma exsertion, which hampers pollination and causes fruit set failure. However, the underlying regulatory mechanisms of the HT-induced stigma exsertion remain largely unknown. Here, we demonstrate that stigma exsertion induced by HT in cultivated tomato is caused by more seriously shortened stamens than pistils, which is different from the stigma exsertion observed in wild tomato species. Under the HT condition, the different responses of pectin, sugar, expansin, and cyclin cause cell wall remodelling and differentially localized cell division and selective cell enlargement, which further determine the lengths of stamens and pistils. In addition, auxin and jasmonate (JA) are implicated in regulating cell division and cell expansion in stamens and pistils, and exogenous JA instead of auxin treatment can effectively rescue tomato stigma exsertion through regulating the JA/COI1 signalling pathway. Our findings provide a better understanding of stigma exsertions under the HT condition in tomato and uncover a new function of JA in improving plant abiotic stress tolerance.
Asunto(s)
Ciclopentanos/metabolismo , Flores/fisiología , Oxilipinas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal , Solanum lycopersicum/metabolismo , Flores/crecimiento & desarrollo , Fructosa/metabolismo , Glucosa/metabolismo , Calor , Ácidos Indolacéticos/metabolismo , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/fisiología , Pectinas/metabolismo , Polinización , Reacción en Cadena en Tiempo Real de la Polimerasa , Autofecundación , Sacarosa/metabolismoRESUMEN
Mitogen-activated protein kinases (MAPKs) regulate diverse aspects of plant growth. However, their potential role in reproductive development remains elusive. Here, we discovered an unique role of SlMPK20, a plant-specific group D MAPK, in pollen development in tomato. RNAi-mediated suppression of SlMPK20 or its knockout using CRISPR/Cas9 significantly reduced or completely abolished pollen viability, respectively, with no effects on maternal fertility. Cell biology and gene expression analyses established that SlMPK20 exerts its role specifically at the uni-to-binucleate transition during microgametogenesis. This assertion is based on the findings that the transgenic pollen was largely arrested at the binucleate stage with the appearance of subcellular abnormality at the middle uninucleate microspore stage; and SlMPK20 mRNA and SlMPK20-GUS signals were localized in the tetrads, uninuclear microspores and binuclear pollen grains but not in microspore mother cells or mature pollen grains. Transcriptomic and proteomic analyses revealed that knockout of SlMPK20 significantly reduced the expression of a large number of genes controlling sugar and auxin metabolism and signaling in anthers. Finally, protein-protein interaction assays identified SlMYB32 as a putative target protein of SlMPK20. We conclude that SlMPK20 specifically regulates post-meiotic pollen development through modulating sugar and auxin metabolism and signaling.
Asunto(s)
Ácidos Indolacéticos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal , Solanum lycopersicum/enzimología , Azúcares/metabolismo , Gametogénesis en la Planta/fisiología , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/fisiología , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteómica , Interferencia de ARNRESUMEN
BACKGROUND: Autogamy in cultivated tomato varieties is a derived trait from wild type tomato plants, which are mostly allogamous. However, environmental stresses can cause morphological defects in tomato flowers and hinder autogamy. Under elevated temperatures, tomato plants usually exhibit the phenotype of stigma exsertion, with severely hindered self-pollination and fruit setting, whereas the inherent mechanism of stigma exsertion have been hitherto unknown. Numerous small RNAs (sRNAs) have been shown to play significant roles in plant development and stress responses, however, none of them have been studied with respect to stamen and pistil development under high-temperature conditions. We investigated the associations between stigma exsertion and small RNAs using high-throughput sequencing technology and molecular biology approaches. RESULTS: Sixteen sRNA libraries of Micro-Tom were constructed from plants stamen and pistil samples and sequenced after 2 d and 12 d of exposure to heat stress, respectively, from which a total of 110 known and 84 novel miRNAs were identified. Under heat stress conditions, 34 known and 35 novel miRNAs were differentially expressed in stamens, and 20 known and 10 novel miRNAs were differentially expressed in pistils. GO and KEGG pathway analysis showed that the predicted target genes of differentially expressed miRNAs were significantly enriched in metabolic pathways in both stamen and pistil libraries. Potential miRNA-target cleavage cascades that correlated with the regulation of stigma exsertion under heat stress conditions were found and validated through qRT-PCR and RLM-5' RACE. CONCLUSION: Overall, a global spectrum of known and novel miRNAs involved in tomato stigma exsertion and induced by high temperatures were identified using high-throughput sequencing and molecular biology approaches, laying a foundation for revealing the miRNA-mediated regulatory network involved in the development of tomato stamens and pistils under high-temperature conditions.
Asunto(s)
Perfilación de la Expresión Génica , Respuesta al Choque Térmico/genética , MicroARNs/genética , Solanum lycopersicum/genética , Solanum lycopersicum/fisiología , Secuencia de Bases , Flores/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARNRESUMEN
The two-component system (TCS), which comprises histidine kinases (HKs), phosphotransfers (HPs), and response regulator proteins (RRs), plays pivotal roles in regulating plant growth, development, and responses to biotic and abiotic stresses. TCS genes have been comprehensively identified and investigated in various crops but poorly characterized in tomato. In this work, a total of 65 TCS genes consisting of 20 HK(L)s, six HPs, and 39 RRs were identified from tomato genome. The classification, gene structures, conserved domains, chromosome distribution, phylogenetic relationship, gene duplication events, and subcellular localization of the TCS gene family were predicted and analyzed in detail. The amino acid sequences of tomato TCS family members, except those of type-B RRs, are highly conserved. The gene duplication events of the TCS family mainly occurred in the RR family. Furthermore, the expansion of RRs was attributed to both segment and tandem duplication. The subcellular localizations of the selected green fluorescent protein (GFP) fusion proteins exhibited a diverse subcellular targeting, thereby confirming their predicted divergent functionality. The majority of TCS family members showed distinct organ- or development-specific expression patterns. In addition, most of TCS genes were induced by abiotic stresses and exogenous phytohormones. The full elucidation of TCS elements will be helpful for comprehensive analysis of the molecular biology and physiological role of the TCS superfamily.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genoma de Planta , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Estrés Fisiológico , Cromosomas de las Plantas , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Solanum lycopersicum/efectos de los fármacos , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Filogenia , Proteínas de Plantas/genéticaRESUMEN
BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade consists of three types of reversibly phosphorylated kinases, namely, MAPK, MAPK kinase (MAPKK/MEK), and MAPK kinase kinase (MAPKKK/MEKK), playing important roles in plant growth, development, and defense response. The MAPK cascade genes have been investigated in detail in model plants, including Arabidopsis, rice, and tomato, but poorly characterized in cucumber (Cucumis sativus L.), a major popular vegetable in Cucurbitaceae crops, which is highly susceptible to environmental stress and pathogen attack. RESULTS: A genome-wide analysis revealed the presence of at least 14 MAPKs, 6 MAPKKs, and 59 MAPKKKs in the cucumber genome. Phylogenetic analyses classified all the CsMAPK and CsMAPKK genes into four groups, whereas the CsMAPKKK genes were grouped into the MEKK, RAF, and ZIK subfamilies. The expansion of these three gene families was mainly contributed by segmental duplication events. Furthermore, the ratios of non-synonymous substitution rates (Ka) and synonymous substitution rates (Ks) implied that the duplicated gene pairs had experienced strong purifying selection. Real-time PCR analysis demonstrated that some MAPK, MAPKK and MAPKKK genes are preferentially expressed in specific organs or tissues. Moreover, the expression levels of most of these genes significantly changed under heat, cold, drought, and Pseudoperonospora cubensis treatments. Exposure to abscisic acid and jasmonic acid markedly affected the expression levels of these genes, thereby implying that they may play important roles in the plant hormone network. CONCLUSION: A comprehensive genome-wide analysis of gene structure, chromosomal distribution, and evolutionary relationship of MAPK cascade genes in cucumber are present here. Further expression analysis revealed that these genes were involved in important signaling pathways for biotic and abiotic stress responses in cucumber, as well as the response to plant hormones. Our first systematic description of the MAPK, MAPKK, and MAPKKK families in cucumber will help to elucidate their biological roles in plant.
Asunto(s)
Cucumis sativus/crecimiento & desarrollo , Cucumis sativus/genética , Perfilación de la Expresión Génica , Genómica , Familia de Multigenes/genética , Proteínas Serina-Treonina Quinasas/genética , Estrés Fisiológico/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Mapeo Cromosómico , Secuencia Conservada , Cucumis sativus/citología , Cucumis sativus/fisiología , Evolución Molecular , Duplicación de Gen , Genoma de Planta/genética , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/química , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Filogenia , Reguladores del Crecimiento de las Plantas/farmacología , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Alineación de Secuencia , Transcripción GenéticaRESUMEN
The CRISPR/Cas system has emerged as a versatile platform for sequence-specific genome engineering in plants. Beyond genome editing, CRISPR/Cas systems, based on nuclease-deficient Cas9 (dCas9), have been repurposed as an RNA-guided platform for transcriptional regulation. CRISPR activation (CRISPRa) represents a novel gain-of-function (GOF) strategy, conferring robust over-expression of the target gene within its native chromosomal context. The CRISPRa systems enable precise, scalable, and robust RNA-guided transcription activation, holding great potential for a variety of fundamental and translational research. In this chapter, we provide a step-by-step guide for efficient gene activation in Arabidopsis based on a highly robust CRISPRa system, CRISPR-Act3.0. We present detailed procedures on the sgRNA design, CRISPR-Act3.0 system construction, Agrobacterium-mediated transformation of Arabidopsis using the floral dip method, and identification of desired transgenic plants.
Asunto(s)
Arabidopsis , Arabidopsis/genética , ARN Guía de Sistemas CRISPR-Cas , Agrobacterium , Sistemas CRISPR-Cas/genética , ARNRESUMEN
Genome editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized plant breeding through targeted genome and transcriptome modifications. However, accurate insertion of large DNA cargoes remains challenging. Recently, Sun and colleagues introduced PrimeRoot, a groundbreaking technology that enables precise and targeted integration of large DNA cargoes into plant genomes with remarkable efficiency and accuracy.
Asunto(s)
Fitomejoramiento , Plantas , Plantas/genética , Edición Génica , Genoma de Planta/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Sistemas CRISPR-Cas/genéticaRESUMEN
CRISPR-Cas nuclease systems, base editors, and CRISPR activation have greatly advanced plant genome engineering. However, the combinatorial approaches for multiplexed orthogonal genome editing and transcriptional regulation were previously unexploited in plants. We have recently established a single Cas9 protein-based CRISPR-Combo platform, enabling efficient multiplexed orthogonal genome editing (double-strand break-mediated genome editing or base editing) and transcriptional activation in plants via engineering the single guide RNA (sgRNA) structure. Here, we provide step-by-step instructions for constructing CRISPR-Combo systems for speed breeding of transgene-free, genome-edited Arabidopsis plants and enhancing rice regeneration with more heritable targeted mutations in a hormone-free manner. We also provide guidance on designing efficient sgRNA, Agrobacterium-mediated transformation of Arabidopsis and rice, rice regeneration without exogenous plant hormones, gene editing evaluation and visual identification of transgene-free Arabidopsis plants with high editing activity. With the use of this protocol, it takes ~2 weeks to establish the CRISPR-Combo systems, 4 months to obtain transgene-free genome-edited Arabidopsis plants and 4 months to obtain rice plants with enrichment of heritable targeted mutations by hormone-free tissue culture.
Asunto(s)
Arabidopsis , Edición Génica , Edición Génica/métodos , Activación Transcripcional , Arabidopsis/genética , Fitomejoramiento , Sistemas CRISPR-Cas/genética , Genoma de Planta , Plantas Modificadas Genéticamente/genéticaRESUMEN
Next-generation sequencing technologies have revolutionized our ability to read sequence information at the genome and transcriptome levels in a high-throughput manner. However, genetic screening at a large or genomic scale remains challenging in plants. Recently, the RNA-guided CRISPR-Cas nucleases have been optimized for high-throughput functional genomic screens combined with guide RNA (gRNA) libraries in plants. This approach has shown great promise in facilitating genetic screening, directed evolution, and quantitative trait engineering. However, this technology is still in its infancy. In this short review, we describe the recent progress in gRNA library-based CRISPR screens in plants. We provide a critical assessment of the current approaches and emerging delivery methods for CRISPR screens. We also highlight the challenges and present future perspectives on CRISPR screens in plants.
Asunto(s)
Sistemas CRISPR-Cas , Genoma , Sistemas CRISPR-Cas/genética , Biblioteca de Genes , Genómica/métodos , ARN Guía de Sistemas CRISPR-CasRESUMEN
The CRISPR-associated Cas12b system is the third most efficient CRISPR tool for targeted genome editing in plants after Cas9 and Cas12a. Although the genome editing ability of AaCas12b has been previously investigated in rice, its off-target effects in plants are largely not known. In this study, we first engineered single-guide RNA (sgRNA) complexes with various RNA scaffolds to enhance editing frequency. We targeted EPIDERMAL PATTERNING FACTOR LIKE 9 (OsEPFL9) and GRAIN SIZE 3 (OsGS3) genes with GTTG and ATTC protospacer adjacent motifs, respectively. The use of two Alicyclobacillus acidoterrestris scaffolds (Aac and Aa1.2) significantly increased the frequency of targeted mutagenesis. Next, we performed whole-genome sequencing (WGS) of stably transformed T0 rice plants to assess off-target mutations. WGS analysis revealed background mutations in both coding and noncoding regions with no evidence of sgRNA-dependent off-target activity in edited genomes. We also showed Mendelian segregation of insertion and deletion (indel) mutations in T1 generation. In conclusion, both Aac and Aa1.2 scaffolds provided precise and heritable genome editing in rice.
Asunto(s)
Edición Génica , Oryza , Sistemas CRISPR-Cas/genética , Oryza/genética , Genoma de Planta , MutaciónRESUMEN
Ralstonia solanacearum, a bacterial plant pathogen, poses a significant threat to tomato (Solanum lycopersicum) production through destructive wilt disease. While noncoding RNA has emerged as a crucial regulator in plant disease, its specific involvement in tomato bacterial wilt remains limited. Here, we conducted a comprehensive analysis of the transcriptional landscape, encompassing both mRNAs and noncoding RNAs, in a tomato resistant line ('ZRS_7') and a susceptible line ('HTY_9') upon R. solanacearum inoculation using high-throughput RNA sequencing. Differential expression (DE) analysis revealed significant alterations in 7506 mRNAs, 997 lncRNAs, and 69 miRNAs between 'ZRS_7' and 'HTY_9' after pathogen exposure. Notably, 4548 mRNAs, 367 lncRNAs, and 26 miRNAs exhibited genotype-specific responses to R. solanacearum inoculation. GO and KEGG pathway analyses unveiled the potential involvement of noncoding RNAs in the response to bacterial wilt disease, targeting receptor-like kinases, cell wall-related genes, glutamate decarboxylases, and other key pathways. Furthermore, we constructed a comprehensive competing endogenous RNA (ceRNA) network incorporating 13 DE-miRNAs, 30 DE-lncRNAs, and 127 DEGs, providing insights into their potential contributions to the response against bacterial inoculation. Importantly, the characterization of possible endogenous target mimics (eTMs) of Sly-miR482e-3p via VIGS technology demonstrated the significant impact of eTM482e-3p-1 silencing on tomato's sensitivity to R. solanacearum. These findings support the existence of an eTM482e-3p-1-Sly-miR482e-3p-NBS-LRRs network in regulating tomato's response to the pathogen. Collectively, our findings shed light on the intricate interactions among lncRNAs, miRNAs, and mRNAs as underlying factors in conferring resistance to R. solanacearum in tomato.
Asunto(s)
MicroARNs , ARN Largo no Codificante , Ralstonia solanacearum , Solanum lycopersicum , Ralstonia solanacearum/genética , Ralstonia solanacearum/metabolismo , Solanum lycopersicum/genética , Transcriptoma , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiologíaRESUMEN
CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)-mediated genome editing has revolutionized fundamental research and plant breeding. Beyond gene editing, CRISPR/Cas systems have been repurposed as a platform for programmable transcriptional regulation. Catalytically inactive Cas variants (dCas), when fused with transcriptional activation domains, allow for specific activation of any target gene in the genome without inducing DNA double-strand breaks. CRISPR activation enables simultaneous activation of multiple genes, holding great promise in the identification of gene regulatory networks and rewiring of metabolic pathways. Here, we describe a simple protocol for constructing a dCas9-mediated multiplexed gene activation system based on the CRISPR-Act3.0 system. The resulting vectors are tested in rice protoplasts. © 2022 Wiley Periodicals LLC. Basic Protocol 1: sgRNA design and construction of CRISPR-Act3.0 vectors for multiplexed gene activation Basic Protocol 2: Determining the activation efficiency of CRISPR-Act3.0 vectors using rice protoplasts.
Asunto(s)
Edición Génica , Fitomejoramiento , Sistemas CRISPR-Cas/genética , Plantas/genética , Activación TranscripcionalRESUMEN
CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.
Asunto(s)
Sistemas CRISPR-Cas , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Sistemas CRISPR-Cas/genética , ADN/genética , Endonucleasas/genética , Edición Génica , Humanos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genéticaRESUMEN
CRISPR-Cas9, its derived base editors and CRISPR activation systems have greatly aided genome engineering in plants. However, these systems are mostly used separately, leaving their combinational potential largely untapped. Here we develop a versatile CRISPR-Combo platform, based on a single Cas9 protein, for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants. We showcase the powerful applications of CRISPR-Combo for boosting plant genome editing. First, CRISPR-Combo is used to shorten the plant life cycle and reduce the efforts in screening transgene-free genome-edited plants by activation of a florigen gene in Arabidopsis. Next, we demonstrate accelerated regeneration and propagation of genome-edited plants by activation of morphogenic genes in poplar. Furthermore, we apply CRISPR-Combo to achieve rice regeneration without exogenous plant hormones, which is established as a new method to predominately enrich heritable targeted mutations. In conclusion, CRISPR-Combo is a versatile genome engineering tool with promising applications in crop breeding.
Asunto(s)
Arabidopsis , Edición Génica , Arabidopsis/genética , Sistemas CRISPR-Cas , Genoma de Planta , Fitomejoramiento , Plantas Modificadas Genéticamente/genéticaRESUMEN
CRISPR/Cas systems have been widely used for genome engineering in many plant species. However, their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only a few reports on the application of the CRISPR/Cas9 system in pear have been documented, and have shown very low editing efficiency. Here we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand the targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taking these results together, we have built a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear.
RESUMEN
The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated) system-mediated precise genome editing has revolutionized genome engineering due to ease of use and versatility of multiplexing. Catalytically inactivated Cas variants (dCas) further expand the usefulness of the CRISPR/Cas system for genetics studies and translational research without inducing DNA double-strand breaks. Fusion of diverse effector domains to dCas proteins empowers the CRISPR/dCas system as a multifunctional platform for gene expression regulation, epigenetic regulation and sequence-specific imaging. In this short review, we summarize the recent advances of CRISPR/dCas-mediated transcriptional activation and repression, and epigenetic modifications. We also highlight the future directions and broader applications of the CRISPR/dCas systems in plants.