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Crop Genome Editing

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 (31 December 2022) | Viewed by 62020

Special Issue Editor

Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843, USA
Interests: plant genetics; genomics; gene editing; crop improvement

Special Issue Information

Dear Colleagues,

Since 2012, innovations in CRISPR/Cas-mediated genome editing have been transforming a wide range of fields, from basic biological research to human therapeutics. One field that is ideally suited to genome editing applications is crop improvement due to the power of targeted mutations for novel trait development. The past few decades have seen tremendous advances in understanding the genetic control of key agronomic traits through QTL and GWAS mapping, whole genome sequencing, and candidate gene analysis. CRISPR/Cas technologies now promise to rapidly accelerate functional gene validation of candidate genes, while at the same time providing an approach to precisely modify key genes controlling beneficial traits in crop species. Moreover, updated regulatory policies in many countries are now facilitating the commercialization of non-transgenic gene-edited cultivars. However, there are still bottlenecks that need to be addressed before high-throughput CRISPR/Cas-based genome editing becomes routine across the major crops, including the need to improve plant transformation efficiency, speed up in vitro tissue culture processes, and optimize precise allele modification, such as by making homology-dependent repair (HDR) more efficient. Once these challenges are overcome, a new era of crop genome editing will simultaneously accelerate the functional validation of key genes while enabling plant breeding to develop the next generation of improved crops to provide more sustainable agriculture and healthier foods for the future.

This Special Issue, “Crop Genome Editing”, will include research topics and review articles that address how CRISPR/Cas-mediated genome editing can be applied toward plant genetic research and crop improvement. Original research articles describing novel crop genome editing techniques, approaches, and innovations are encouraged, as well as case studies where genome editing has been used for gene characterization and validation, novel trait development, or genetic dissection of key traits for crop improvement, including stress tolerance, disease resistance, and improved quality and nutrition.

Dr. Endang Septiningsih
Guest Editor

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Keywords

  • CRISPR/Cas9
  • crop improvement
  • functional gene validation
  • genome editing
  • molecular genetics

Published Papers (17 papers)

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13 pages, 2639 KiB  
Article
Mutation of GmIPK1 Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds
by Ji Hyeon Song, Gilok Shin, Hye Jeong Kim, Saet Buyl Lee, Ju Yeon Moon, Jae Cheol Jeong, Hong-Kyu Choi, In Ah Kim, Hyeon Jin Song, Cha Young Kim and Young-Soo Chung
Int. J. Mol. Sci. 2022, 23(18), 10583; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231810583 - 13 Sep 2022
Cited by 15 | Viewed by 2186
Abstract
Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP5) [...] Read more.
Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP5) to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in soybean (Glycine max L.). In this study, for developing soybean with low PA levels, we attempted to edit the GmIPK1 gene using the CRISPR/Cas9 system to introduce mutations into the GmIPK1 gene with guide RNAs in soybean (cv. Kwangankong). The GmIPK1 gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean Gmipk1 gene-edited lines were obtained in the T0 generation at editing frequencies of 0.1–84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1–9 nucleotides and insertions of 1 nucleotide in several soybean lines (T0). Finally, we confirmed two sgRNA-4 Gmipk1 gene-edited homozygote soybean T1 plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean Gmipk1 gene-edited lines indicated a reduction in PA content in soybean T2 seeds but did not show any defects in plant growth and seed development. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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13 pages, 3514 KiB  
Article
Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity
by Sudip Biswas, Aya Bridgeland, Samra Irum, Michael J. Thomson and Endang M. Septiningsih
Int. J. Mol. Sci. 2022, 23(17), 9809; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23179809 - 29 Aug 2022
Cited by 16 | Viewed by 2625
Abstract
Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and [...] Read more.
Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and a prime editing guide RNA (pegRNA). In addition, prime editing has a wider range of editing types than base editing and can produce nearly all types of edits. Although prime editing was first established in human cells, it has recently been applied to plants. As a relatively new technique, optimization will be needed to increase the editing efficiency in different crops. In this study, we successfully edited a mutant GFP in rice, peanut, chickpea, and cowpea protoplasts. In rice, up to 16 times higher editing efficiency was achieved with a dual pegRNA than the single pegRNA containing vectors. Edited-mutant GFP protoplasts have also been obtained in peanut, chickpea, and cowpea after transformation with the dual pegRNA vectors, albeit with much lower editing efficiency than in rice, ranging from 0.2% to 0.5%. These initial results promise to expedite the application of prime editing in legume breeding programs to accelerate crop improvement. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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11 pages, 2180 KiB  
Article
Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools
by Dongchang Zeng, Zhiye Zheng, Yuxin Liu, Taoli Liu, Tie Li, Jianhong Liu, Qiyu Luo, Yang Xue, Shengting Li, Nan Chai, Suize Yu, Xianrong Xie, Yao-Guang Liu and Qinlong Zhu
Int. J. Mol. Sci. 2022, 23(14), 7990; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23147990 - 20 Jul 2022
Cited by 12 | Viewed by 2182
Abstract
CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) [...] Read more.
CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5′-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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8 pages, 1403 KiB  
Communication
Application of CRISPR/CasΦ2 System for Genome Editing in Plants
by Qinan Cai, Dongmei Guo, Yujun Cao, Yuan Li, Rui Ma and Wenping Liu
Int. J. Mol. Sci. 2022, 23(10), 5755; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23105755 - 20 May 2022
Cited by 6 | Viewed by 1812
Abstract
CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the [...] Read more.
CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg2+ irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg2+ irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg2+ ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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8 pages, 1413 KiB  
Communication
Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis
by M. Moniruzzaman, Yun Zhong, Zhifeng Huang and Guangyan Zhong
Int. J. Mol. Sci. 2022, 23(9), 4889; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23094889 - 28 Apr 2022
Cited by 1 | Viewed by 1705
Abstract
Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of [...] Read more.
Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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16 pages, 2934 KiB  
Article
CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers
by Stephany Toinga-Villafuerte, Maria Isabel Vales, Joseph M. Awika and Keerti S. Rathore
Int. J. Mol. Sci. 2022, 23(9), 4640; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23094640 - 22 Apr 2022
Cited by 22 | Viewed by 7022
Abstract
Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and [...] Read more.
Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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15 pages, 4456 KiB  
Article
Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds
by Tia Dunbar, Nikolaos Tsakirpaloglou, Endang M. Septiningsih and Michael J. Thomson
Int. J. Mol. Sci. 2022, 23(8), 4081; https://doi.org/10.3390/ijms23084081 - 07 Apr 2022
Cited by 14 | Viewed by 2980
Abstract
CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to [...] Read more.
CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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13 pages, 3439 KiB  
Article
A Model to Incorporate the bHLH Transcription Factor OsIRO3 within the Rice Iron Homeostasis Regulatory Network
by Oscar Carey-Fung, Martin O’Brien, Jesse T. Beasley and Alexander A. T. Johnson
Int. J. Mol. Sci. 2022, 23(3), 1635; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031635 - 31 Jan 2022
Cited by 7 | Viewed by 2887
Abstract
Iron (Fe) homeostasis in plants is governed by a complex network of regulatory elements and transcription factors (TFs), as both Fe toxicity and deficiency negatively impact plant growth and physiology. The Fe homeostasis network is well characterized in Arabidopsis thaliana and remains poorly [...] Read more.
Iron (Fe) homeostasis in plants is governed by a complex network of regulatory elements and transcription factors (TFs), as both Fe toxicity and deficiency negatively impact plant growth and physiology. The Fe homeostasis network is well characterized in Arabidopsis thaliana and remains poorly understood in monocotyledon species such as rice (Oryza sativa L.). Recent investigation of the rice Fe homeostasis network revealed OsIRO3, a basic Helix–Loop–Helix (bHLH) TF as a putative negative regulator of genes involved in Fe uptake, transport, and storage. We employed CRISPR-Cas9 gene editing to target the OsIRO3 coding sequence and generate two independent T-DNA-free, loss-of-function iro3 mutants in rice cv. Nipponbare. The iro3 mutant plants had similar phenotype under nutrient-sufficient conditions and had stunted growth under Fe-deficient conditions, relative to a T-DNA free, wild-type control (WT). Under Fe deficiency, iro3 mutant shoots had reduced expression of Fe chelator biosynthetic genes (OsNAS1, OsNAS2, and OsNAAT1) and upregulated expression of an Fe transporter gene (OsYSL15), relative to WT shoots. We place our results in the context of the existing literature and generate a model describing the role of OsIRO3 in rice Fe homeostasis and reinforce the essential function of OsIRO3 in the rice Fe deficiency response. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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11 pages, 3245 KiB  
Article
An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice
by Jiantao Tan, Yaxi Wang, Shuifu Chen, Zhansheng Lin, Yanchang Zhao, Yang Xue, Yuyu Luo, Yao-Guang Liu and Qinlong Zhu
Int. J. Mol. Sci. 2022, 23(3), 1588; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031588 - 29 Jan 2022
Cited by 5 | Viewed by 2371
Abstract
In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, [...] Read more.
In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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13 pages, 3989 KiB  
Article
Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2
by Sudip Biswas, Nancy J. Wahl, Michael J. Thomson, John M. Cason, Bill F. McCutchen and Endang M. Septiningsih
Int. J. Mol. Sci. 2022, 23(2), 837; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23020837 - 13 Jan 2022
Cited by 18 | Viewed by 4014
Abstract
The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and [...] Read more.
The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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12 pages, 2104 KiB  
Article
A New RING Finger Protein, PLANT ARCHITECTURE and GRAIN NUMBER 1, Affects Plant Architecture and Grain Yield in Rice
by Peiwen Yan, Yu Zhu, Ying Wang, Fuying Ma, Dengyong Lan, Fuan Niu, Shiqing Dong, Xinwei Zhang, Jian Hu, Siwen Liu, Tao Guo, Xiaoyun Xin, Shiyong Zhang, Jinshui Yang, Liming Cao and Xiaojin Luo
Int. J. Mol. Sci. 2022, 23(2), 824; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23020824 - 13 Jan 2022
Cited by 10 | Viewed by 1993
Abstract
Developing methods for increasing the biomass and improving the plant architecture is important for crop improvement. We herein describe a gene belonging to the RING_Ubox (RING (Really Interesting New Gene) finger domain and U-box domain) superfamily, PLANT ARCHITECTURE and GRAIN NUMBER 1 [...] Read more.
Developing methods for increasing the biomass and improving the plant architecture is important for crop improvement. We herein describe a gene belonging to the RING_Ubox (RING (Really Interesting New Gene) finger domain and U-box domain) superfamily, PLANT ARCHITECTURE and GRAIN NUMBER 1 (PAGN1), which regulates the number of grains per panicle, the plant height, and the number of tillers. We used the CRISPR/Cas9 system to introduce loss-of-function mutations to OsPAGN1. Compared with the control plants, the resulting pagn1 mutant plants had a higher grain yield because of increases in the plant height and in the number of tillers and grains per panicle. Thus, OsPAGN1 may be useful for the genetic improvement of plant architecture and yield. An examination of evolutionary relationships revealed that OsPAGN1 is highly conserved in rice. We demonstrated that OsPAGN1 can interact directly with OsCNR10 (CELL NUMBER REGULATOR10), which negatively regulates the number of rice grains per panicle. A transcriptome analysis indicated that silencing OsPAGN1 affects the levels of active cytokinins in rice. Therefore, our findings have clarified the OsPAGN1 functions related to rice growth and grain development. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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20 pages, 3520 KiB  
Article
A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9
by María H. Guzmán-López, Miriam Marín-Sanz, Susana Sánchez-León and Francisco Barro
Int. J. Mol. Sci. 2021, 22(23), 13076; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222313076 - 03 Dec 2021
Cited by 4 | Viewed by 2734
Abstract
The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, [...] Read more.
The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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21 pages, 8816 KiB  
Article
Molecular and Functional Analysis of U-box E3 Ubiquitin Ligase Gene Family in Rice (Oryza sativa)
by Me-Sun Kim, Kwon-Kyoo Kang and Yong-Gu Cho
Int. J. Mol. Sci. 2021, 22(21), 12088; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222112088 - 08 Nov 2021
Cited by 11 | Viewed by 3030
Abstract
Proteins encoded by U-box type ubiquitin ligase (PUB) genes in rice are known to play an important role in plant responses to abiotic and biotic stresses. Functional analysis has revealed a detailed molecular mechanism involving PUB proteins in relation to abiotic and biotic [...] Read more.
Proteins encoded by U-box type ubiquitin ligase (PUB) genes in rice are known to play an important role in plant responses to abiotic and biotic stresses. Functional analysis has revealed a detailed molecular mechanism involving PUB proteins in relation to abiotic and biotic stresses. In this study, characteristics of 77 OsPUB genes in rice were identified. Systematic and comprehensive analyses of the OsPUB gene family were then performed, including analysis of conserved domains, phylogenetic relationships, gene structure, chromosome location, cis-acting elements, and expression patterns. Through transcriptome analysis, we confirmed that 16 OsPUB genes show similar expression patterns in drought stress and blast infection response pathways. Numerous cis-acting elements were found in promoter sequences of 16 OsPUB genes, indicating that the OsPUB genes might be involved in complex regulatory networks to control hormones, stress responses, and cellular development. We performed qRT-PCR on 16 OsPUB genes under drought stress and blast infection to further identify the reliability of transcriptome and cis-element analysis data. It was confirmed that the expression pattern was similar to RNA-sequencing analysis results. The transcription of OsPUB under various stress conditions indicates that the PUB gene might have various functions in the responses of rice to abiotic and biotic stresses. Taken together, these results indicate that the genome-wide analysis of OsPUB genes can provide a solid basis for the functional analysis of U-box E3 ubiquitin ligase genes. The molecular information of the U-box E3 ubiquitin ligase gene family in rice, including gene expression patterns and cis-acting regulatory elements, could be useful for future crop breeding programs by genome editing. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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16 pages, 1576 KiB  
Article
Optimizing Agrobacterium-Mediated Transformation and CRISPR-Cas9 Gene Editing in the tropical japonica Rice Variety Presidio
by Marco Molina-Risco, Oneida Ibarra, Mayra Faion-Molina, Backki Kim, Endang M. Septiningsih and Michael J. Thomson
Int. J. Mol. Sci. 2021, 22(20), 10909; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222010909 - 09 Oct 2021
Cited by 15 | Viewed by 3708
Abstract
Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols [...] Read more.
Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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21 pages, 1236 KiB  
Review
CRISPR/Cas9 Technology and Its Utility for Crop Improvement
by Hua Liu, Wendan Chen, Yushu Li, Lei Sun, Yuhong Chai, Haixia Chen, Haochen Nie and Conglin Huang
Int. J. Mol. Sci. 2022, 23(18), 10442; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231810442 - 09 Sep 2022
Cited by 12 | Viewed by 4292
Abstract
The rapid growth of the global population has resulted in a considerable increase in the demand for food crops. However, traditional crop breeding methods will not be able to satisfy the worldwide demand for food in the future. New gene-editing technologies, the most [...] Read more.
The rapid growth of the global population has resulted in a considerable increase in the demand for food crops. However, traditional crop breeding methods will not be able to satisfy the worldwide demand for food in the future. New gene-editing technologies, the most widely used of which is CRISPR/Cas9, may enable the rapid improvement of crop traits. Specifically, CRISPR/Cas9 genome-editing technology involves the use of a guide RNA and a Cas9 protein that can cleave the genome at specific loci. Due to its simplicity and efficiency, the CRISPR/Cas9 system has rapidly become the most widely used tool for editing animal and plant genomes. It is ideal for modifying the traits of many plants, including food crops, and for creating new germplasm materials. In this review, the development of the CRISPR/Cas9 system, the underlying mechanism, and examples of its use for editing genes in important crops are discussed. Furthermore, certain limitations of the CRISPR/Cas9 system and potential solutions are described. This article will provide researchers with important information regarding the use of CRISPR/Cas9 gene-editing technology for crop improvement, plant breeding, and gene functional analyses. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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16 pages, 1529 KiB  
Review
Control of Bacterial Diseases of Banana Using CRISPR/Cas-Based Gene Editing
by Leena Tripathi, Valentine O. Ntui and Jaindra N. Tripathi
Int. J. Mol. Sci. 2022, 23(7), 3619; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23073619 - 25 Mar 2022
Cited by 14 | Viewed by 8639
Abstract
Banana is an important staple food crop and a source of income for smallholder farmers in about 150 tropical and sub-tropical countries. Several bacterial diseases, such as banana Xanthomonas wilt (BXW), blood, and moko disease, cause substantial impacts on banana production. There is [...] Read more.
Banana is an important staple food crop and a source of income for smallholder farmers in about 150 tropical and sub-tropical countries. Several bacterial diseases, such as banana Xanthomonas wilt (BXW), blood, and moko disease, cause substantial impacts on banana production. There is a vast yield gap in the production of bananas in regions where bacterial pathogens and several other pathogens and pests are present together in the same field. BXW disease caused by Xanthomonas campestris pv. musacearum is reported to be the most destructive banana disease in East Africa. The disease affects all the banana varieties grown in the region. Only the wild-type diploid banana, Musa balbisiana, is resistant to BXW disease. Developing disease-resistant varieties of bananas is one of the most effective strategies to manage diseases. Recent advances in CRISPR/Cas-based gene editing techniques can accelerate banana improvement. Some progress has been made to create resistance against bacterial pathogens using CRISPR/Cas9-mediated gene editing by knocking out the disease-causing susceptibility (S) genes or activating the expression of the plant defense genes. A synopsis of recent advancements and perspectives on the application of gene editing for the control of bacterial wilt diseases are presented in this article. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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20 pages, 1240 KiB  
Review
Engineering Properties of Sweet Potato Starch for Industrial Applications by Biotechnological Techniques including Genome Editing
by Ruiqing Lyu, Sulaiman Ahmed, Weijuan Fan, Jun Yang, Xiaoyun Wu, Wenzhi Zhou, Peng Zhang, Ling Yuan and Hongxia Wang
Int. J. Mol. Sci. 2021, 22(17), 9533; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22179533 - 02 Sep 2021
Cited by 16 | Viewed by 5357
Abstract
Sweet potato (Ipomoea batatas) is one of the largest food crops in the world. Due to its abundance of starch, sweet potato is a valuable ingredient in food derivatives, dietary supplements, and industrial raw materials. In addition, due to its ability [...] Read more.
Sweet potato (Ipomoea batatas) is one of the largest food crops in the world. Due to its abundance of starch, sweet potato is a valuable ingredient in food derivatives, dietary supplements, and industrial raw materials. In addition, due to its ability to adapt to a wide range of harsh climate and soil conditions, sweet potato is a crop that copes well with the environmental stresses caused by climate change. However, due to the complexity of the sweet potato genome and the long breeding cycle, our ability to modify sweet potato starch is limited. In this review, we cover the recent development in sweet potato breeding, understanding of starch properties, and the progress in sweet potato genomics. We describe the applicational values of sweet potato starch in food, industrial products, and biofuel, in addition to the effects of starch properties in different industrial applications. We also explore the possibility of manipulating starch properties through biotechnological means, such as the CRISPR/Cas-based genome editing. The ability to target the genome with precision provides new opportunities for reducing breeding time, increasing yield, and optimizing the starch properties of sweet potatoes. Full article
(This article belongs to the Special Issue Crop Genome Editing)
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