The application of CRISPR/Cas technologies to Brassica crops: current progress and future perspectives

Li, Jun; Yu, Xiaoxiao; Zhang, Chao; Li, Na; Zhao, Jianjun

doi:10.1007/s42994-022-00076-3

Cited by 15 publications

(11 citation statements)

References 121 publications

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“…Therefore, CRISPR/Cas systems provide dynamic and effective immunity against invading genetic elements. Sequence-specific RNA–DNA recognition and cleavage by Cas nucleases make CRISPR/Cas systems useful as programmable genome editing tools [ 28 ].…”

Section: Crispr/cas: a Useful Genome Editing Toolmentioning

confidence: 99%

“…Then, Cas9 utilizes two distinct nuclease domains, HNH and RuvC-like, to cleave both strands of the target DNA, generating sequence-specific DSBs. This triggers two DNA repair systems, nonhomologous end-joining (NHEJ) and homology-directed repair (HDR) [ 28 ]. NHEJ, which is prevalent in most cells, is an error-prone pathway.…”

Section: Crispr/cas: a Useful Genome Editing Toolmentioning

confidence: 99%

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Virus-Induced Gene Editing and Its Applications in Plants

Zhang

Liu

et al. 2022

IJMS

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CRISPR/Cas-based genome editing technologies, which allow the precise manipulation of plant genomes, have revolutionized plant science and enabled the creation of germplasms with beneficial traits. In order to apply these technologies, CRISPR/Cas reagents must be delivered into plant cells; however, this is limited by tissue culture challenges. Recently, viral vectors have been used to deliver CRISPR/Cas reagents into plant cells. Virus-induced genome editing (VIGE) has emerged as a powerful method with several advantages, including high editing efficiency and a simplified process for generating gene-edited DNA-free plants. Here, we briefly describe CRISPR/Cas-based genome editing. We then focus on VIGE systems and the types of viruses used currently for CRISPR/Cas9 cassette delivery and genome editing. We also highlight recent applications of and advances in VIGE in plants. Finally, we discuss the challenges and potential for VIGE in plants.

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Section: Crispr/cas: a Useful Genome Editing Toolmentioning

confidence: 99%

Section: Crispr/cas: a Useful Genome Editing Toolmentioning

confidence: 99%

Virus-Induced Gene Editing and Its Applications in Plants

Zhang

Liu

et al. 2022

IJMS

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“…The recent sequencing and assembly of the cotton genome has opened the door to the systematic study of gene function [2][3][4]. The emergence of modern biotechnologies, including single-cell RNA sequencing (scRNAseq) and genome editing, will promote the development of breeding strategies and basic research in cotton [5][6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis

Zhang

Liu²,

Fu³

et al. 2023

Plant Methods

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Background Cotton has tremendous economic value worldwide; however, its allopolyploid nature and time-consuming transformation methods have hampered the development of cotton functional genomics. The protoplast system has proven to be an important and versatile tool for functional genomics, tissue-specific marker gene identification, tracking developmental trajectories, and genome editing in plants. Nevertheless, the isolation of abundant viable protoplasts suitable for single-cell RNA sequencing (scRNA-seq) and genome editing remains a challenge in cotton. Results We established an efficient transient gene expression system using protoplasts isolated from cotton taproots. The system enables the isolation of large numbers of viable protoplasts and uses an optimized PEG-mediated transfection protocol. The highest yield (3.55 × 105/g) and viability (93.3%) of protoplasts were obtained from cotton roots grown in hydroponics for 72 h. The protoplasts isolated were suitable for scRNA-seq. The highest transfection efficiency (80%) was achieved when protoplasts were isolated as described above and transfected with 20 μg of plasmid for 20 min in a solution containing 200 mM Ca2+. Our protoplast-based transient expression system is suitable for various applications, including validation the efficiency of CRISPR vectors, protein subcellular localization analysis, and protein–protein interaction studies. Conclusions The protoplast isolation and transfection protocol developed in this study is stable, versatile, and time-saving. It will accelerate functional genomics and molecular breeding in cotton.

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“…Most were conducted in Brassica oleracea or B. napus to improve commercially important agronomic traits or nutritional values such as seed oil, carotenoids, or glucosinolate contents . Most of these studies used CRISPR/CRISPR-associated protein 9 (Cas9) to generate insertions/deletions (InDels) in single or multiple genes [ 11 ]. Only a few studies have reported modifying genes using CRISPR/Cas9 in B. rapa .…”

Section: Introductionmentioning

confidence: 99%

Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage

Park,

Lee,

Song

et al. 2023

Horticulture Research

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Flavonols are the major class of flavonoids of green Chinese cabbage (Brassica rapa subsp. pekinensis). The B. rapa genome harbors seven flavonol synthase genes (BrFLSs), but they have not been functionally characterized. Here, transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage. Among them, only BrFLS1 showed major FLS activity and additional flavanone 3β-hydroxylase (F3H) activity, while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities. We generated BrFLS1-knockout (BrFLS1-KO) Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T1 generation, which were further advanced to the T2 generation showing normal phenotype. UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 plants, while dihydroflavonol glycosides accumulated concomitantly at corresponding levels. Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were up-regulated in the BrFLS1-KO plants. In accordance, total phenolic contents were slightly enhanced in the BrFLS1-KO plants, which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage. Phenotypic surveys revealed that the brfls1 Chinese cabbages showed normal head formation and reproductive phenotypes, but subtle morphological changes in their heads were observed. In addition, their seedlings were susceptible to osmotic stress compared to the controls, suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa. In this study, we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.

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The application of CRISPR/Cas technologies to Brassica crops: current progress and future perspectives

Cited by 15 publications

References 121 publications

Virus-Induced Gene Editing and Its Applications in Plants

Virus-Induced Gene Editing and Its Applications in Plants

Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis

Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage

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