Prospects for Knockout of MYB60, a Transcriptional Repressor of Anthocyanin Biosynthesis, in Brassicaceae Plants by Genome Editing

The Brassicaceae plant family contains many economically important crops such as Brassica napus L., Brassica rapa L., Brassica oleracea L., Brassica juncea L., Eruca sativa Mill., Camelina sativa L. and Raphanus sativus L. Insufficient data on the genetic regulation of agronomic traits in these species complicates the editing of their genomes. In recent years, the attention of the academic community has been drawn to anthocyanin hyperaccumulation. This trait is not only beneficial for human health, but can also increase plant resistance to stress. MYB transcription factors are the main regulators of flavonoid biosynthesis in plants. Some of them are well studied in Arabidopsis thaliana. The AtMYB60 gene is a transcriptional repressor of anthocyanin biosynthesis, and it also negatively impacts plant responses to drought stress. Myb60 is one of the least studied transcription factors with similar functions in Brassicaceae. There is a high degree of homology between predicted MYB60 genes of A. thaliana and related plant species. However, functions of these homologous genes have never been studied. Gene knockout by CRISPR/Cas technology remains the easiest way to perform genome editing in order to discover the role of individual plant genes. Disruption of genes acting as negative regulators of anthocyanin biosynthesis could result in color staining of plant tissues and an increase in stress tolerance. In the present study, we investigated the AtMYB60 gene and its homologs in Brassicaceae plants and suggested universal gRNAs to knockout these genes. Keywords: CRISPR, Brassicaceae, MYB60, knockout, anthocyanin

Download Full-text

Editing of the MYB genes in Brassica napus as a method to increase anthocyanin pigmentation and stress tolerance

E3S Web of Conferences ◽

10.1051/e3sconf/202022404022 ◽

2020 ◽

Vol 224 ◽

pp. 04022

Author(s):

E V Mikhaylova ◽

M. Y. Shein ◽

A. Y. Artyukhin ◽

A S Sukhareva ◽

M. A. Panfilova ◽

...

Keyword(s):

Transcription Factors ◽

Brassica Napus ◽

Genome Editing ◽

Anthocyanin Biosynthesis ◽

Phytopathogenic Fungi ◽

Anthocyanin Pigmentation ◽

Dna Binding Domains ◽

Binding Domains ◽

Myb Genes ◽

Genetic Constructs

Anthocyanin hyperaccumulation is an important agricultural trait, associated with resistance to abiotic stress, pests, phytopathogenic fungi and bacterial diseases. B. napus with increased anthocyanin pigmentation can be generated by genome editing. Many transcription factors of the MYB family are involved in stress response and anthocyanin biosynthesis. Genes AtMYB60, AtCPC and AtMYBL2 are negative regulators of anthocyanin biosynthesis in Arabidopsis, therefore the knockout of these genes can result in increased anthocyanin pigmentation. gRNA spacers were synthesized to target the orthologs of these genes, identified in Brassica napus. Resulting genetic constructs were introduced to the plant tissues by agroinfiltration. Transient expression of gRNAs targeting DNA-binding domains of MYB transcription factors along with Cas9 nuclease successfully promoted anthocyanin hyperaccumulation. These genetic constructs can be used for genome editing and production of new colored and stress tolerant varieties of oilseed rape.

Download Full-text

Diverse Systems for Efficient Sequence Insertion and Replacement in Precise Plant Genome Editing

BioDesign Research ◽

10.34133/2020/8659064 ◽

2020 ◽

Vol 2020 ◽

pp. 1-4

Author(s):

Yingxiao Zhang ◽

Yiping Qi

Keyword(s):

Genome Editing ◽

Genetic Variants ◽

Agronomic Traits ◽

Gene Knockout ◽

Plant Genome ◽

Chemically Modified ◽

Limited Success ◽

Efficient Sequence

CRISPR-mediated genome editing has been widely applied in plants to make uncomplicated genomic modifications including gene knockout and base changes. However, the introduction of many genetic variants related to valuable agronomic traits requires complex and precise DNA changes. Different CRISPR systems have been developed to achieve efficient sequence insertion and replacement but with limited success. A recent study has significantly improved NHEJ- and HDR-mediated sequence insertion and replacement using chemically modified donor templates. Together with other newly developed precise editing systems, such as prime editing and CRISPR-associated transposases, these technologies will provide new avenues to further the plant genome editing field.

Download Full-text

CRISPR/Cas9-Mediated Multiplex Genome Editing of JAGGED Gene in Brassica napus L.

Biomolecules ◽

10.3390/biom9110725 ◽

2019 ◽

Vol 9 (11) ◽

pp. 725 ◽

Cited By ~ 2

Author(s):

Qamar U Zaman ◽

Wen Chu ◽

Mengyu Hao ◽

Yuqin Shi ◽

Mengdan Sun ◽

...

Keyword(s):

Brassica Napus ◽

Genome Editing ◽

Gene Knockout ◽

Cylindrical Body ◽

High Yield ◽

Wild Type ◽

Brassica Napus L ◽

Pod Shattering ◽

Undifferentiated Cells ◽

Key Factor

Pod shattering resistance is an essential component to achieving a high yield, which is a substantial objective in polyploid rapeseed cultivation. Previous studies have suggested that the Arabidopsis JAGGED (JAG) gene is a key factor implicated in the regulatory web of dehiscence fruit. However, its role in controlling pod shattering resistance in oilseed rape is still unknown. In this study, multiplex genome editing was carried out by the CRISPR/Cas9 system on five homoeologs (BnJAG.A02, BnJAG.C02, BnJAG.C06, BnJAG.A07, and BnJAG.A08) of the JAG gene. Knockout mutagenesis of all homoeologs drastically affected the development of the lateral organs in organizing pod shape and size. The cylindrical body of the pod comprised a number of undifferentiated cells like a callus, without distinctive valves, replum, septum, and valve margins. Pseudoseeds were produced, which were divided into two halves with an incomplete layer of cells (probably septum) that separated the undifferentiated cells. These mutants were not capable of generating any productive seeds for further generations. However, one mutant line was identified in which only a BnJAG.A08-NUB-Like paralog of the JAG gene was mutated. Knockout mutagenesis in BnJAG.A08-NUB gene caused significant changes in the pod dehiscence zone. The replum region of the mutant was increased to a great extent, resulting in enlarged cell size, bumpy fruit, and reduced length compared with the wild type. A higher replum–valve joint area may have increased the resistance to pod shattering by ~2-fold in JAG mutants compared with wild type. Our results offer a basis for understanding variations in Brassica napus fruit by mutating JAG genes and providing a way forward for other Brassicaceae species.

Download Full-text

Unravelling the Complex Interplay of Transcription Factors Orchestrating Seed Oil Content in Brassica napus L.

International Journal of Molecular Sciences ◽

10.3390/ijms22031033 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1033

Author(s):

Abirami Rajavel ◽

Selina Klees ◽

Johanna-Sophie Schlüter ◽

Hendrik Bertram ◽

Kun Lu ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Brassica Napus ◽

Oil Content ◽

Seed Oil ◽

Seed Oil Content ◽

Complex Interplay ◽

Brassica Napus L ◽

Data Set ◽

Developmental Switches

Transcription factors (TFs) and their complex interplay are essential for directing specific genetic programs, such as responses to environmental stresses, tissue development, or cell differentiation by regulating gene expression. Knowledge regarding TF–TF cooperations could be promising in gaining insight into the developmental switches between the cultivars of Brassica napus L., namely Zhongshuang11 (ZS11), a double-low accession with high-oil- content, and Zhongyou821 (ZY821), a double-high accession with low-oil-content. In this regard, we analysed a time series RNA-seq data set of seed tissue from both of the cultivars by mainly focusing on the monotonically expressed genes (MEGs). The consideration of the MEGs enables the capturing of multi-stage progression processes that are orchestrated by the cooperative TFs and, thus, facilitates the understanding of the molecular mechanisms determining seed oil content. Our findings show that TF families, such as NAC, MYB, DOF, GATA, and HD-ZIP are highly involved in the seed developmental process. Particularly, their preferential partner choices as well as changes in their gene expression profiles seem to be strongly associated with the differentiation of the oil content between the two cultivars. These findings are essential in enhancing our understanding of the genetic programs in both cultivars and developing novel hypotheses for further experimental studies.

Download Full-text

Genome-Wide Mining of MYB Transcription Factors in the Anthocyanin Biosynthesis Pathway of Gossypium Hirsutum

Biochemical Genetics ◽

10.1007/s10528-021-10027-0 ◽

2021 ◽

Author(s):

Yingjie Zhu ◽

Ying Bao

Keyword(s):

Transcription Factors ◽

Gossypium Hirsutum ◽

Anthocyanin Biosynthesis ◽

Biosynthesis Pathway ◽

Myb Transcription Factors ◽

Genome Wide

Download Full-text

Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

aBIOTECH ◽

10.1007/s42994-021-00042-5 ◽

2021 ◽

Author(s):

Jun Li ◽

Yan Li ◽

Ligeng Ma

Keyword(s):

Functional Genomics ◽

Genome Editing ◽

Functional Annotation ◽

Genome Engineering ◽

Agronomic Traits ◽

Wheat Breeding ◽

Breeding Programs ◽

Base Editing ◽

The World ◽

World Food Security

AbstractCommon wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

Download Full-text

Comparative transcriptome analysis reveals key genes associated with pigmentation in radish (Raphanus sativus L.) skin and flesh

Scientific Reports ◽

10.1038/s41598-021-90633-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jifang Zhang ◽

Jian Zhao ◽

Qunyun Tan ◽

Xiaojun Qiu ◽

Shiyong Mei

Keyword(s):

Transcription Factors ◽

Raphanus Sativus ◽

Regulatory Networks ◽

Anthocyanin Biosynthesis ◽

Comparative Transcriptome ◽

Structural Genes ◽

Key Genes ◽

Transcript Profiles ◽

Species Specific ◽

Anthocyanin Biosynthetic Genes

AbstractRadish (Raphanus sativus) is an important vegetable worldwide that exhibits different flesh and skin colors. The anthocyanins responsible for the red and purple coloring in radishes possess nutritional value and pharmaceutical potential. To explore the structural and regulatory networks related to anthocyanin biosynthesis and identify key genes, we performed comparative transcriptome analyses of the skin and flesh of six colored radish accessions. The transcript profiles showed that each accession had a species-specific transcript profile. For radish pigmentation accumulation, the expression levels of anthocyanin biosynthetic genes (RsTT4, RsC4H, RsTT7, RsCCOAMT, RsDFR, and RsLDOX) were significantly upregulated in the red- and purple-colored accessions, but were downregulated or absent in the white and black accessions. The correlation test, combined with metabolome (PCC > 0.95), revealed five structural genes (RsTT4, RsDFR, RsCCOAMT, RsF3H, and RsBG8L) and three transcription factors (RsTT8-1, RsTT8-2, and RsPAR1) to be significantly correlated with flavonoids in the skin of the taproot. Four structural genes (RsBG8L, RsDFR, RsCCOAMT, and RsLDOX) and nine transcription factors (RsTT8-1, RsTT8-2, RsMYB24L, RsbHLH57, RsPAR2L, RsbHLH113L, RsOGR3L, RsMYB24, and RsMYB34L) were found to be significantly correlated with metabolites in the flesh of the taproot. This study provides a foundation for future studies on the gene functions and genetic diversity of radish pigmentation and should aid in the cultivation of new valuable radish varieties.

Download Full-text

An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽

10.1128/mspheredirect.00149-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 33

Author(s):

Namkha Nguyen ◽

Morgan M. F. Quail ◽

Aaron D. Hernday

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Fungal Pathogen ◽

Gene Knockout ◽

Strain Engineering ◽

Molecular Genetic Analysis ◽

Content Type ◽

Highly Efficient ◽

Discovery Research ◽

Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

Download Full-text