Updated in October 2019
Fruit crops, including apple, orange, grape, banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops, including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including dCas9‐base‐editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene‐free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumer‐friendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.
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 be 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. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.
Auxin is an important plant hormone that is essential for growth and development due to its effects on organogenesis, morphogenesis, tropisms, and apical dominance. The functional diversity of auxin highlights the importance of its biosynthesis, transport, and associated responses. In this study, we show that a NAC transcription factor, ANAC092 (also named AtNAC2 and ORESARA1), known to positively regulate leaf senescence and contribute to abiotic stress responses, also affects primary root development. Plants overexpressing ANAC092 had altered root meristem lengths and shorter primary roots compared with the wild‐type control. Additionally, expression of the proANAC092::GUS was strongly induced by indole‐3‐acetic acid. Quantitative real‐time RT‐PCR (qRT‐PCR) analysis revealed that the YUCCA2, PIN, and ARF expression levels were downregulated in ANAC092‐overexpressing plants. Moreover, yeast one‐hybrid and chromatin immunoprecipitation assays confirmed that ANAC092 binds to the promoters of AUXIN RESPONSE FACTOR 8 (ARF8) and PIN‐FORMED 4 (PIN4). Furthermore, a dual‐luciferase assay indicated that ANAC092 decreases ARF8 and PIN4 promoter activities. We also applied a CRISPR/Cas9 system to mutate ANAC092. The roots of three of the analyzed mutants were longer than normal. Collectively, our findings indicate that ANAC092 negatively affects root development by controlling the auxin pathway.
During the establishment of rhizobia-legume symbiosis, the cytokinin receptor
LHK1 (Lotus Histidine Kinase 1) is essential for nodule formation. However, the
mechanism by which cytokinin signaling regulates symbiosis remains largely
unknown. In this study, an LHK1-interacting protein, LjCZF1, was identified and
further characterized. LjCZF1 is a C3HC4-type RING finger protein that is highly
conserved in plants. LjCZF1 specifically interacted with LHK1 in yeast
two-hybrid, in vitro pull-down and co-immunoprecipitation assays
conducted in tobacco. Phosphomimetic mutation of the potential threonine (T167D)
phosphorylation site enhanced the interaction between LjCZF1 and LHK1, whereas
phosphorylation mutation (T167A) eliminated this interaction. Transcript
abundance of LjCZF1 was up-regulated significantly after inoculation
with rhizobia. The LORE1 insertion mutant and clustered regularly
interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein
9-mediated knockout mutant Lotus japonicus plants demonstrated
significantly reduced number of infection threads and nodules. In contrast,
plants over-expressing LjCZF1 exhibited increased numbers of infection
threads and nodules. Collectively, these data support the notion that LjCZF1 is
a positive regulator of symbiotic nodulation, possibly through interaction with
Hydrogen peroxide (H2O2) is generated in many metabolic processes. As a signaling molecule, H2O2 plays important roles in plant growth and development, as well as environmental stress response. In Arabidopsis, there are three catalase genes, CAT1, CAT2, and CAT3. The encoded catalases are predominately peroxisomal proteins and are critical for scavenging H2O2. Since CAT1 and CAT3 are linked on chromosome 1, it has been almost impossible to generate cat1/3 and cat1/2/3 mutants by traditional genetic tools. In this study, we constructed cat1/3 double mutants and cat1/2/3 triple mutants by CRISPR/Cas9 to investigate the role of catalases. The cat1/2/3 triple mutants displayed severe redox disturbance and growth defects under physiological conditions compared with wild‐type and the cat2/3 double mutants. Transcriptome analysis showed a more profound transcriptional response in the cat1/2/3 triple mutants compared to the cat2/3 mutants. These differentially expressed genes are involved in plant growth regulation as well as abiotic and biotic stress responses. In addition, expression of OXI1 (OXIDATIVE SIGNAL INDUCIBLE 1) and several MAPK cascade genes were changed dramatically in the catalase triple mutant, suggesting that H2O2 produced in peroxisomes could serve as a peroxisomal retrograde signal.
Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. A single nucleotide polymorphism in the NRT1.1B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Herein, we precisely replaced the japonica NRT1.1B allele with the indica allele, in just one generation, using CRISPR/Cas9 gene‐editing technology. No additional selective pressure was needed to enrich the precise replacement events. This work demonstrates the feasibility of replacing any genes with elite alleles within one generation, greatly expanding our ability to improve agriculturally important traits.
In rice, amylose content (AC) is controlled by a single dominant Waxy gene. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR‐associated 9 (Cas9) to introduce a loss‐of‐function mutation into the Waxy gene in two widely cultivated elite japonica varieties. Our results show that mutations in the Waxy gene reduce AC and convert the rice into glutinous ones without affecting other desirable agronomic traits, offering an effective and easy strategy to improve glutinosity in elite varieties. Importantly, we successfully removed the transgenes from the progeny. Our study provides an example of generating improved crops with potential for commercialization, by editing a gene of interest directly in elite crop varieties.
We report that a solo single‐guide RNA (sgRNA) seed is capable of guiding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR −associated 9 (CRISRP/Cas9) to simultaneously edit multiple genes AtRPL10A, AtRPL10B and AtRPL10C in Arabidopsis. Our results also demonstrate that it is possible to use CRISPR/Cas9 technology to create AtRPL10 triple mutants which otherwise cannot be generated by conventional genetic crossing. Compared to other conventional multiplex CRISPR/Cas systems, a single sgRNA seed has the advantage of reducing off‐target gene‐editing. Such a gene editing system might be also applicable to modify other homologous genes, or even less‐homologous sequences for multiple gene‐editing in plants and other organisms.
Chloroplast genes are transcribed by the plastid-encoded RNA polymerase (PEP) or nucleus-encoded RNA polymerase. FRUCTOKINASE-LIKE PROTEINS (FLNs) are phosphofructokinase-B (PfkB)-type carbohydrate kinases that act as part of the PEP complex; however, the molecular mechanisms underlying FLN activity in rice remain elusive. Previously, we identified and characterized a heat-stress sensitive albino (hsa1) mutant in rice. Map-based cloning revealed that HSA1 encodes a putative OsFLN2. Here, we further demonstrated that knockdown or knockout of the OsFLN1, a close homolog of HSA1/OsFLN2, considerably inhibits chloroplast biogenesis and the fln1 knockout mutants, created by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associate protein 9, exhibit severe albino phenotype and seedling lethality. Moreover, OsFLN1 localizes to the chloroplast. Yeast two-hybrid, pull-down and bimolecular fluorescence complementation experiments revealed that OsFLN1 and HSA1/OsFLN2 interact with THIOREDOXINZ (OsTRXz) to regulate chloroplast development. In agreement with this, knockout of OsTRXz resulted in a similar albino and seedling lethality phenotype to that of the fln1 mutants. Quantitative reverse transcription polymerase chain reaction and immunoblot analysis revealed that the transcription and translation of PEP-dependent genes were strongly inhibited in fln1 and trxz mutants, indicating that loss of OsFLN1, HSA1/OsFLN2, or OsTRXz function perturbs the stability of the transcriptionally active chromosome complex and PEP activity. These results show that OsFLN1 and HSA1/OsFLN2 contribute to chloroplast biogenesis and plant growth.
Grain yield is one of the most important and complex trait for genetic improvement in crops; it is known to be controlled by a number of genes known as quantitative trait loci (QTLs). In the past decade, many yield-contributing QTLs have been identified in crops. However, it remains unclear whether those QTLs confer the same yield performance in different genetic backgrounds. Here, we performed CRISPR/Cas9-mediated QTL editing in five widely-cultivated rice varieties and revealed that the same QTL can have diverse, even opposing, effects on grain yield in different genetic backgrounds.
Hybrids between the indica and japonica subspecies of rice (Oryza sativa) are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding. © 2017 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.
The transcription factors CBF1/2/3 are reported to play a dominant role in the cold responsive network of Arabidopsis by directly regulating the expression levels of cold responsive (COR) genes. In this study, we obtained CRISPR/Cas9-mediated loss-of-function mutants of cbf1∼3. Over 3,000 COR genes identified by RNA-seq analysis showed a slight but significant change in their expression levels in the mutants compared to the wild-type plants after being treated at 4 °C for 12 h. The C-repeat (CRT) motif (5′-CCGAC-3′) was enriched in promoters of genes that were up-regulated by CBF2 and CBF3 but not in promoters of genes up-regulated by CBF1. These data suggest that CBF2 and CBF3 play a more important role in directing the cold response by regulating different sets of downstream COR genes. More than 2/3 of COR genes were co-regulated by two or three CBFs and were involved mainly in cellular signal transduction and metabolic processes; less than 1/3 of the genes were regulated by one CBF, and those genes up-regulated were enriched in cold-related abiotic stress responses. Our results indicate that CBFs play an important role in the trade-off between cold tolerance and plant growth through the precise regulation of COR genes in the complicated transcriptional network.
CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19−20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTM system and unique EcoRI/XhoI sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user-friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present CRISPR Primer Designer for researchers to design primers for CRISPR applications. The program has a user-friendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRISPR-Cas system-based chromosome imaging system.
Yan M, Zhou SR, Xue HW (2015) CRISPR Primer Designer: Design primers for knockout and chromosome imaging CRISPR-Cas system. J Integr Plant Biol 57: 613–617. doi: 10.1111/jipb.12295
CRISPR/Cas9 uses a guide RNA (gRNA) molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.
Gao Y, Zhao Y (2014) Self‐processing of ribozyme‐ﬂanked RNAs into guide RNAs in vitro and in vivo for CRISPR‐mediated genome editing. J Integr Plant Biol 56: 343–349. doi: 10.1111/jipb.12152