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Genome Editing in Plants

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Plant Sciences".

Deadline for manuscript submissions: closed (1 May 2019) | Viewed by 141205

Special Issue Editor


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Guest Editor
Department of Biology, East Carolina University, Greenville, NC 27858, USA
Interests: genome editing; small regulatory RNAs; molecular genetics; plant biotechnology; abiotic stress; gene expression and regulation
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Genome editing is becoming a powerful tool for gene functional study and also for improving plants for growth and development as well as tolerance to various abiotic and biotic stress. In the past a couple of years, as new Cas are identified and new bioinformatics tools have been developed; this field has been developing quickly. Currently, it has been widely used in many plant species not only for gene functional study but also for crop improvement. In this Special Issue, both reviews and research papers, as well as method and bioinformatics papers, are welcomed.

Prof. Dr. Baohong Zhang
Guest Editor

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Keywords

  • genome editing
  • CRISPR/Cas9
  • plant functional study
  • gene knockout
  • gene regulation
  • plant improvement
  • plant biotechnology

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Published Papers (17 papers)

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Editorial

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3 pages, 166 KiB  
Editorial
CRISPR/Cas9: A Robust Genome-Editing Tool with Versatile Functions and Endless Application
by Baohong Zhang
Int. J. Mol. Sci. 2020, 21(14), 5111; https://doi.org/10.3390/ijms21145111 - 20 Jul 2020
Cited by 7 | Viewed by 3134
Abstract
Since a potential genome editing tool was first recognized in 2012 [...] Full article
(This article belongs to the Special Issue Genome Editing in Plants)

Research

Jump to: Editorial, Review

13 pages, 2814 KiB  
Article
CRISPR/Cas9-Based Mutagenesis of Starch Biosynthetic Genes in Sweet Potato (Ipomoea Batatas) for the Improvement of Starch Quality
by Hongxia Wang, Yinliang Wu, Yandi Zhang, Jun Yang, Weijuan Fan, Hui Zhang, Shanshan Zhao, Ling Yuan and Peng Zhang
Int. J. Mol. Sci. 2019, 20(19), 4702; https://doi.org/10.3390/ijms20194702 - 23 Sep 2019
Cited by 88 | Viewed by 9120
Abstract
CRISPR/Cas9-mediated genome editing is a powerful technology that has been used for the genetic modification of a number of crop species. In order to evaluate the efficacy of CRISPR/Cas9 technology in the root crop, sweet potato (Ipomoea batatas), two starch biosynthetic [...] Read more.
CRISPR/Cas9-mediated genome editing is a powerful technology that has been used for the genetic modification of a number of crop species. In order to evaluate the efficacy of CRISPR/Cas9 technology in the root crop, sweet potato (Ipomoea batatas), two starch biosynthetic pathway genes, IbGBSSI (encoding granule-bound starch synthase I), and IbSBEII (encoding starch branching enzyme II), were targeted in the starch-type cultivar Xushu22 and carotenoid-rich cultivar Taizhong6. I. batatas was transformed using a binary vector, in which the Cas9 gene is driven by the Arabidopsis AtUBQ promoter and the guide RNA is controlled by the Arabidopsis AtU6 promoter. A total of 72 Xushu22 and 35 Taizhong6 transgenic lines were generated and analyzed for mutations. The mutation efficiency was 62–92% with multi-allelic mutations in both cultivars. Most of the mutations were nucleotide substitutions that lead to amino acid changes and, less frequently, stop codons. In addition, short nucleotide insertions or deletions were also found in both IbGBSSI and IbSBEII. Furthermore, a 2658 bp deletion was found in one IbSBEII transgenic line. The total starch contents were not significantly changed in IbGBSSI- and IbSBEII-knockout transgenic lines compared to the wild-type control. However, in the allopolyploid sweet potato, the IbGBSSI-knockout reduced, while the IbSBEII-knockout increased, the amylose percentage. Our results demonstrate that CRISPR/Cas9 technology is an effective tool for the improvement of starch qualities in sweet potato and breeding of polyploid root crops. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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19 pages, 2990 KiB  
Article
Highly Efficient and Heritable Targeted Mutagenesis in Wheat via the Agrobacterium tumefaciens-Mediated CRISPR/Cas9 System
by Shujuan Zhang, Rongzhi Zhang, Jie Gao, Tiantian Gu, Guoqi Song, Wei Li, Dandan Li, Yulian Li and Genying Li
Int. J. Mol. Sci. 2019, 20(17), 4257; https://doi.org/10.3390/ijms20174257 - 30 Aug 2019
Cited by 29 | Viewed by 5636
Abstract
The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we [...] Read more.
The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we demonstrated that the CRISPR/Cas9 system could achieve efficient mutagenesis in five wheat genes via Agrobacterium-mediated transformation of an sgRNA targeting the D genome, an sgRNA targeting both the A and B homologues and three tri-genome guides targeting the editing of all three homologues. High mutation rates and putative homozygous or biallelic mutations were observed in the T0 plants. The targeted mutations could be stably inherited by the next generation, and the editing efficiency of each mutant line increased significantly across generations. The editing types and inheritance of targeted mutagenesis were similar, which were not related to the targeted subgenome number. The presence of Cas9/sgRNA could cause new mutations in subsequent generations, while mutated lines without Cas9/sgRNA could retain the mutation type. Additionally, off-target mutations were not found in sequences that were highly homologous to the selected sgRNA sequences. Overall, the results suggested that CRISPR/Cas9-induced gene editing via Agrobacterium-mediated transformation plays important roles in wheat genome engineering. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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11 pages, 2574 KiB  
Article
H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies
by Tsung-Meng Wu, Jian-Zhi Huang, Hui-Min Oung, Yi-Ting Hsu, Yu-Chang Tsai and Chwan-Yang Hong
Int. J. Mol. Sci. 2019, 20(16), 3885; https://doi.org/10.3390/ijms20163885 - 9 Aug 2019
Cited by 18 | Viewed by 5581
Abstract
Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede [...] Read more.
Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3′-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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14 pages, 899 KiB  
Article
Genetic Structure Analysis of a Collection of Tunisian Durum Wheat Germplasm
by Amine Slim, Luciana Piarulli, Houda Chennaoui Kourda, Mustapha Rouaissi, Cyrine Robbana, Ramzi Chaabane, Domenico Pignone, Cinzia Montemurro and Giacomo Mangini
Int. J. Mol. Sci. 2019, 20(13), 3362; https://doi.org/10.3390/ijms20133362 - 9 Jul 2019
Cited by 25 | Viewed by 4650
Abstract
The Tunisian durum wheat germplasm includes modern cultivars and traditional varieties that are still cultivated in areas where elite cultivars or intensive cultivation systems are not suitable. Within the frame of a collection program of the National Gene Bank of Tunisia (NGBT), durum [...] Read more.
The Tunisian durum wheat germplasm includes modern cultivars and traditional varieties that are still cultivated in areas where elite cultivars or intensive cultivation systems are not suitable. Within the frame of a collection program of the National Gene Bank of Tunisia (NGBT), durum wheat germplasm was collected from different Tunisian agro-ecological zones. The collected samples were studied using simple sequence repeats (SSRs) markers to explore the genetic diversity and evaluate the genetic structure in Tunisian germplasm. The results demonstrated significant diversity in the Tunisian durum wheat germplasm, with clear differentiation between traditional varieties and modern cultivars. The population structure analysis allowed the identification of five subpopulations, two of which appear to be more strongly represented in germplasm collected in central and southern Tunisia, where environmental conditions at critical development phases of the plant are harsher. Moreover these subpopulations are underrepresented in modern varieties, suggesting that traits of adaptation useful for breeding more resilient varieties might be present in central and southern germplasm. Moreover, our results will support, the activity of in situ on farm conservation of Tunisian durum wheat germplasm started by the National Gene Bank of Tunisia along with the ex situ approach. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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15 pages, 2322 KiB  
Article
Functional Analysis of M-Locus Protein Kinase Revealed a Novel Regulatory Mechanism of Self-Incompatibility in Brassica napus L.
by Fang Chen, Yong Yang, Bing Li, Zhiquan Liu, Fawad Khan, Tong Zhang, Guilong Zhou, Jinxing Tu, Jinxiong Shen, Bin Yi, Tingdong Fu, Cheng Dai and Chaozhi Ma
Int. J. Mol. Sci. 2019, 20(13), 3303; https://doi.org/10.3390/ijms20133303 - 5 Jul 2019
Cited by 32 | Viewed by 4736
Abstract
Self-incompatibility (SI) is a widespread mechanism in angiosperms that prevents inbreeding by rejecting self-pollen. However, the regulation of the SI response in Brassica napus is not well understood. Here, we report that the M-locus protein kinase (MLPK) BnaMLPKs, the functional [...] Read more.
Self-incompatibility (SI) is a widespread mechanism in angiosperms that prevents inbreeding by rejecting self-pollen. However, the regulation of the SI response in Brassica napus is not well understood. Here, we report that the M-locus protein kinase (MLPK) BnaMLPKs, the functional homolog of BrMLPKs in Brassica rapa, controls SI in B. napus. We identified four paralogue MLPK genes in B. napus, including BnaA3.MLPK, BnaC3.MLPK, BnaA4.MLPK, and BnaC4.MLPK. Two transcripts of BnaA3.MLPK, BnaA3.MLPKf1 and BnaA3.MLPKf2, were generated by alternative splicing. Tissue expression pattern analysis demonstrated that BnaA3.MLPK, especially BnaA3.MLPKf2, is highly expressed in reproductive organs, particularly in stigmas. We subsequently created RNA-silencing lines and CRISPR/Cas9-induced quadruple mutants of BnaMLPKs in B. napus SI line S-70. Phenotypic analysis revealed that SI response is partially suppressed in RNA-silencing lines and is completely blocked in quadruple mutants. These results indicate the importance of BnaMLPKs in regulating the SI response of B. napus. We found that the expression of SI positive regulators S-locus receptor kinase (SRK) and Arm-Repeat Containing 1 (ARC1) are suppressed in bnmlpk mutant, whereas the self-compatibility (SC) element Glyoxalase I (GLO1) maintained a high expression level. Overall, our findings reveal a new regulatory mechanism of MLPK in the SI of B. napus. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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15 pages, 3871 KiB  
Article
Genome Editing to Integrate Seed Size and Abiotic Stress Tolerance Traits in Arabidopsis Reveals a Role for DPA4 and SOD7 in the Regulation of Inflorescence Architecture
by Siyu Chen, Na Zhang, Qimeng Zhang, Ganghua Zhou, Hainan Tian, Saddam Hussain, Sajjad Ahmed, Tianya Wang and Shucai Wang
Int. J. Mol. Sci. 2019, 20(11), 2695; https://doi.org/10.3390/ijms20112695 - 31 May 2019
Cited by 34 | Viewed by 4435
Abstract
Both seed size and abiotic stress tolerance are important agronomic traits in crops. In Arabidopsis, two closely related transcription repressors DPA4 (Development-Related PcG Target in the APEX4)/NGAL3 and SOD7 (Suppressor of da1-1)/NGAL2 (NGATHA-like protein) function redundantly to regulate seed size, which was [...] Read more.
Both seed size and abiotic stress tolerance are important agronomic traits in crops. In Arabidopsis, two closely related transcription repressors DPA4 (Development-Related PcG Target in the APEX4)/NGAL3 and SOD7 (Suppressor of da1-1)/NGAL2 (NGATHA-like protein) function redundantly to regulate seed size, which was increased in the dpa4 sod7 double mutants. Whereas ABA-induced transcription repressors (AITRs) are involved in the regulation of ABA signaling and abiotic stress tolerance, Arabidopsis aitr2 aitr5 aitr6 (aitr256) triple mutant showed enhanced tolerance to drought and salt. Here we performed CRISPR/Cas9 genome editing to disrupt DPA4 and SOD7 in aitr256 mutant, trying to integrate seed size and abiotic stress tolerance traits in Arabidopsis, and also to examine whether DPA4 and SOD7 may regulate other aspects of plant growth and development. Indeed, seed size was increased in the dpa4 sod7 aitr256 quintuple mutants, and enhanced tolerance to drought was observed in the mutants. In addition, we found that shoot branching was affected in the dpa4 sod7 aitr256 mutants. The mutant plants failed to produce secondary branches, and flowers/siliques were distributed irregularly on the main stems of the plants. Floral organ number and fertility were also affected in the dpa4 sod7 aitr256 mutant plants. To examine if these phenotypes were dependent on loss-of-function of AITRs, dpa4 sod7 double mutants were generated in Col wild type background, and we found that the dpa4 sod7 mutant plants showed a phenotype similar to the dpa4 sod7 aitr256 quintuple mutants. Taken together, our results indicate that the integration of seed size and abiotic stress tolerance traits by CRISPR/Cas9 editing was successful, and our results also revealed a role of DPA4 and SOD7 in the regulation of inflorescence architecture in Arabidopsis. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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13 pages, 5712 KiB  
Article
Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9
by Jie Ji, Chunyang Zhang, Zhongfeng Sun, Longlong Wang, Deqiang Duanmu and Qiuling Fan
Int. J. Mol. Sci. 2019, 20(10), 2471; https://doi.org/10.3390/ijms20102471 - 19 May 2019
Cited by 90 | Viewed by 7641
Abstract
Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea [...] Read more.
Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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23 pages, 5074 KiB  
Article
Comparative Transcriptome Analysis of Developing Seeds and Silique Wall Reveals Dynamic Transcription Networks for Effective Oil Production in Brassica napus L.
by Muhammad Shahid, Guangqin Cai, Feng Zu, Qing Zhao, Muhammad Uzair Qasim, Yueyun Hong, Chuchuan Fan and Yongming Zhou
Int. J. Mol. Sci. 2019, 20(8), 1982; https://doi.org/10.3390/ijms20081982 - 23 Apr 2019
Cited by 43 | Viewed by 6095
Abstract
Vegetable oil is an essential constituent of the human diet and renewable raw material for industrial applications. Enhancing oil production by increasing seed oil content in oil crops is the most viable, environmentally friendly, and sustainable approach to meet the continuous demand for [...] Read more.
Vegetable oil is an essential constituent of the human diet and renewable raw material for industrial applications. Enhancing oil production by increasing seed oil content in oil crops is the most viable, environmentally friendly, and sustainable approach to meet the continuous demand for the supply of vegetable oil globally. An in-depth understanding of the gene networks involved in oil biosynthesis during seed development is a prerequisite for breeding high-oil-content varieties. Rapeseed (Brassica napus) is one of the most important oil crops cultivated on multiple continents, contributing more than 15% of the world’s edible oil supply. To understand the phasic nature of oil biosynthesis and the dynamic regulation of key pathways for effective oil accumulation in B. napus, comparative transcriptomic profiling was performed with developing seeds and silique wall (SW) tissues of two contrasting inbred lines with ~13% difference in seed oil content. Differentially expressed genes (DEGs) between high- and low-oil content lines were identified across six key developmental stages, and gene enrichment analysis revealed that genes related to photosynthesis, metabolism, carbohydrates, lipids, phytohormones, transporters, and triacylglycerol and fatty acid synthesis tended to be upregulated in the high-oil-content line. Differentially regulated DEG patterns were revealed for the control of metabolite and photosynthate production in SW and oil biosynthesis and accumulation in seeds. Quantitative assays of carbohydrates and hormones during seed development together with gene expression profiling of relevant pathways revealed their fundamental effects on effective oil accumulation. Our results thus provide insights into the molecular basis of high seed oil content (SOC) and a new direction for developing high-SOC rapeseed and other oil crops. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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13 pages, 1708 KiB  
Article
Efficient Editing of the Nuclear APT Reporter Gene in Chlamydomonas reinhardtii via Expression of a CRISPR-Cas9 Module
by Daniel Guzmán-Zapata, José M. Sandoval-Vargas, Karla S. Macedo-Osorio, Edgar Salgado-Manjarrez, José L. Castrejón-Flores, María del Carmen Oliver-Salvador, Noé V. Durán-Figueroa, Fabien Nogué and Jesús A. Badillo-Corona
Int. J. Mol. Sci. 2019, 20(5), 1247; https://doi.org/10.3390/ijms20051247 - 12 Mar 2019
Cited by 41 | Viewed by 7357
Abstract
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that [...] Read more.
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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18 pages, 4759 KiB  
Article
Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory
by Guillaume Bernard, David Gagneul, Harmony Alves Dos Santos, Audrey Etienne, Jean-Louis Hilbert and Caroline Rambaud
Int. J. Mol. Sci. 2019, 20(5), 1155; https://doi.org/10.3390/ijms20051155 - 6 Mar 2019
Cited by 46 | Viewed by 7632
Abstract
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene [...] Read more.
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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10 pages, 1516 KiB  
Communication
Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor
by Florian Veillet, Laura Perrot, Laura Chauvin, Marie-Paule Kermarrec, Anouchka Guyon-Debast, Jean-Eric Chauvin, Fabien Nogué and Marianne Mazier
Int. J. Mol. Sci. 2019, 20(2), 402; https://doi.org/10.3390/ijms20020402 - 18 Jan 2019
Cited by 248 | Viewed by 21432
Abstract
Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived [...] Read more.
Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the acetolactate synthase (ALS) gene in tomato and potato by a CBE using Agrobacterium-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the ALS with other gene(s), leading to transgene-free plants harboring new traits of interest. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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13 pages, 2709 KiB  
Article
CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean
by Yupeng Cai, Li Chen, Shi Sun, Cunxiang Wu, Weiwei Yao, Bingjun Jiang, Tianfu Han and Wensheng Hou
Int. J. Mol. Sci. 2018, 19(12), 3835; https://doi.org/10.3390/ijms19123835 - 1 Dec 2018
Cited by 75 | Viewed by 8645
Abstract
At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of [...] Read more.
At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 ‘transgene-free’ homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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13 pages, 4058 KiB  
Article
Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System
by Shouhong Zhu, Xiuli Yu, Yanjun Li, Yuqiang Sun, Qianhao Zhu and Jie Sun
Int. J. Mol. Sci. 2018, 19(10), 3000; https://doi.org/10.3390/ijms19103000 - 1 Oct 2018
Cited by 30 | Viewed by 5471
Abstract
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene [...] Read more.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers. Based on sequence analysis of the target site in the 10 transgenic cottons containing CRISPR/Cas9, we found that the mutation frequencies of GhALARP-A and GhALARP-D target sites were 71.4–100% and 92.9–100%, respectively. The most common editing event was deletion, but deletion together with large insertion was also observed. Mosaic mutation editing events were detected in most transgenic plants. No off-target mutation event was detected in any the 15 predicted sites analyzed. This study provided mutants for further study of the function of GhALARP in cotton fiber development. Our results further demonstrated the feasibility of use of CRISPR/Cas9 as a targeted mutagenesis tool in cotton, and provided an efficient tool for targeted mutagenesis and functional genomics in cotton. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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Review

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19 pages, 2460 KiB  
Review
Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants
by Zahra Hajiahmadi, Ali Movahedi, Hui Wei, Dawei Li, Yasin Orooji, Honghua Ruan and Qiang Zhuge
Int. J. Mol. Sci. 2019, 20(15), 3719; https://doi.org/10.3390/ijms20153719 - 30 Jul 2019
Cited by 59 | Viewed by 9233
Abstract
The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, [...] Read more.
The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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33 pages, 1204 KiB  
Review
Genome Editing in Agriculture: Technical and Practical Considerations
by Julia Jansing, Andreas Schiermeyer, Stefan Schillberg, Rainer Fischer and Luisa Bortesi
Int. J. Mol. Sci. 2019, 20(12), 2888; https://doi.org/10.3390/ijms20122888 - 13 Jun 2019
Cited by 36 | Viewed by 10603
Abstract
The advent of precise genome-editing tools has revolutionized the way we create new plant varieties. Three groups of tools are now available, classified according to their mechanism of action: Programmable sequence-specific nucleases, base-editing enzymes, and oligonucleotides. The corresponding techniques not only lead to [...] Read more.
The advent of precise genome-editing tools has revolutionized the way we create new plant varieties. Three groups of tools are now available, classified according to their mechanism of action: Programmable sequence-specific nucleases, base-editing enzymes, and oligonucleotides. The corresponding techniques not only lead to different outcomes, but also have implications for the public acceptance and regulatory approval of genome-edited plants. Despite the high efficiency and precision of the tools, there are still major bottlenecks in the generation of new and improved varieties, including the efficient delivery of the genome-editing reagents, the selection of desired events, and the regeneration of intact plants. In this review, we evaluate current delivery and regeneration methods, discuss their suitability for important crop species, and consider the practical aspects of applying the different genome-editing techniques in agriculture. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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18 pages, 2916 KiB  
Review
Applications of the CRISPR/Cas9 System for Rice Grain Quality Improvement: Perspectives and Opportunities
by Sajid Fiaz, Shakeel Ahmad, Mehmood Ali Noor, Xiukang Wang, Afifa Younas, Aamir Riaz, Adeel Riaz and Fahad Ali
Int. J. Mol. Sci. 2019, 20(4), 888; https://doi.org/10.3390/ijms20040888 - 19 Feb 2019
Cited by 104 | Viewed by 18078
Abstract
Grain quality improvement is a key target for rice breeders, along with yield. It is a multigenic trait that is simultaneously influenced by many factors. Over the past few decades, breeding for semi-dwarf cultivars and hybrids has significantly contributed to the attainment of [...] Read more.
Grain quality improvement is a key target for rice breeders, along with yield. It is a multigenic trait that is simultaneously influenced by many factors. Over the past few decades, breeding for semi-dwarf cultivars and hybrids has significantly contributed to the attainment of high yield demands but reduced grain quality, which thus needs the attention of researchers. The availability of rice genome sequences has facilitated gene discovery, targeted mutagenesis, and revealed functional aspects of rice grain quality attributes. Some success has been achieved through the application of molecular markers to understand the genetic mechanisms for better rice grain quality; however, researchers have opted for novel strategies. Genomic alteration employing genome editing technologies (GETs) like clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for reverse genetics has opened new avenues of research in the life sciences, including for rice grain quality improvement. Currently, CRISPR/Cas9 technology is widely used by researchers for genome editing to achieve the desired biological objectives, because of its simple targeting. Over the past few years many genes that are related to various aspects of rice grain quality have been successfully edited via CRISPR/Cas9 technology. Interestingly, studies on functional genomics at larger scales have become possible because of the availability of GETs. In this review, we discuss the progress made in rice by employing the CRISPR/Cas9 editing system and its eminent applications. We also elaborate possible future avenues of research with this system, and our understanding regarding the biological mechanism of rice grain quality improvement. Full article
(This article belongs to the Special Issue Genome Editing in Plants)
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