An Improved CRISPR/Cas9 System for Genome Editing in Populus by Using Mannopine Synthase (MAS) Promoter

Gene editing technology in woody plants has great potential for understanding gene function, and altering traits affecting economically and ecologically important traits. Gene editing applications in woody species require a high genome editing efficiency due to the difficulty during transformation and complexities resulting from gene redundancy. In this study, we used poplar 84K (Populus alba × P. glandulosa), which is a model hybrid for studying wood formation and growth. We developed a new CRISPR/Cas9 system to edit multiple genes simultaneously. Using this system, we successfully knocked out multiple targets of the PHYTOENE DESATURASE 8 in poplar. We found the mutation rate of our CRISPR/Cas9 system is higher (67.5%) than existing reports in woody trees. We further improved the mutation rate up to 75% at editing sites through the usage of the mannopine synthase (MAS) promoter to drive Cas9. The MAS-CRISPR/Cas9 is an improved genome-editing tool for woody plants with a higher efficiency and a higher mutation rate than currently available technologies.

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Efficient Multiplex Genome Editing Tools identified by Protoplast Technology in Phalaenopsis

10.1101/2020.09.29.315200 ◽

2020 ◽

Author(s):

Keke Xia ◽

Dengwei Zhang ◽

Guangyu Liu ◽

Xiaojing Xu ◽

Yong Yang ◽

...

Keyword(s):

Genome Editing ◽

Mutation Rate ◽

Gene Function ◽

Large Scale ◽

Ornamental Plants ◽

Knockout Mutant ◽

Pol Ii ◽

Structure System ◽

Breeding Research ◽

Target Sites

AbstractPhalaenopsis orchids are popular ornamental plants worldwide. The application of the efficient multiplex genome editing tools in Phalaenopsis, will greatly accelerate the development of orchid gene function and breeding research. In this study, we establish a fast and convenient Phalaenopsis protoplast platform for the identification of functional genome editing tools. Two multiplex genome editing tools, PTG-Cas9 (PTG, polycistronic tRNA gRNA) system and PTGm-Cas9 (PTG-Cas9 system with modified sgRNA structure) system are designed to edit PDS gene of commercial Phalaenopsis ST166 at four target sites. We find that both PTG-Cas9 and PTGm-Cas9 system are functional in Phalaenopsis, and the PTGm-Cas9 system with modified sgRNA has a higher editing efficiency than PTG-Cas9 system. Further, we design another multiplex genome editing tool, termed as DPII-Cpf1 system (dual Pol II promoter to drive the expression of Cpf1 endonuclease and crRNA), to edit PDS gene of Phalaenopsis at four target sites likewise. All the four targets are efficiently edited by DPII-Cpf1 system, and the total mutation rate is about 3 times higher than that of PTGm-Cas9 system. Taken together, using the Phalaenopsis protoplast platform, we successfully establish two efficient multiplex genome editing tools for Phalaenopsis research, PTGm-Cas9 and DPII-Cpf1. The multiplex genome editing tools established in this study have great application potentials in efficiently constructing large-scale knockout mutant libraries of orchid and speeding up orchid precise breeding.

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A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis

International Journal of Molecular Sciences ◽

10.3390/ijms19123925 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3925 ◽

Cited By ~ 12

Author(s):

Zhengyan Feng ◽

Zhengjing Zhang ◽

Kai Hua ◽

Xifeng Gao ◽

Yanfei Mao ◽

...

Keyword(s):

Cell Division ◽

Genome Editing ◽

Plant Species ◽

Mutation Rate ◽

Gene Editing ◽

Mutation Rates ◽

35S Promoter ◽

Ubiquitin Promoter ◽

Homozygous Mutations ◽

Low Efficiency

The CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In Arabidopsis, constitutive promoters usually result in a low efficiency of heritable mutation in the T1 generation. In this work, CRISPR/Cas9 gene editing efficiencies using different promoters to drive Cas9 expression were evaluated. Expression of Cas9 under the constitutive CaMV 35S promoter resulted in a 2.3% mutation rate in T1 plants and failed to produce homozygous mutations in the T1 and T2 generations. In contrast, expression of Cas9 under two cell division-specific promoters, YAO and CDC45, produced mutation rates of 80.9% to 100% in the T1 generation with nonchimeric mutations in the T1 (4.4–10%) and T2 (32.5–46.1%) generations. The pCDC45 promoter was used to modify a previously reported multiplex CRISPR/Cas9 system, replacing the original constitutive ubiquitin promoter. The multi-pCDC45-Cas9 system produced higher mutation efficiencies than the multi-pUBQ-Cas9 system in the T1 generation (60.17% vs. 43.71%) as well as higher efficiency of heritable mutations (11.30% vs. 4.31%). Sextuple T2 homozygous mutants were identified from a construct targeting seven individual loci. Our results demonstrate the advantage of using cell division promoters for CRISPR/Cas9 gene editing applications in Arabidopsis, especially in multiplex applications.

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Highly efficient multiplex genome editing in dicot plants using improved CRISPR/Cas systems

10.1101/2022.01.10.475635 ◽

2022 ◽

Author(s):

Bingjie Li ◽

Yun Shang ◽

Lixianqiu Wang ◽

Jing Lv ◽

Fengjiao Wang ◽

...

Keyword(s):

Life Span ◽

Genome Editing ◽

Gene Function ◽

Gene Editing ◽

Gene Promoter ◽

Highly Efficient ◽

Target Sites

CRISPR/Cas9-mediated gene editing provides a powerful tool for dissecting gene function and improving important traits in crops. However, there are still persisting challenges to obtain high homozygous/bi-allelic (ho/bi) mutations in dicot plants. Here, we develop an improved CRISPR/Cas9 system harboring a calreticulin-like gene promoter, which can boost targeted mutations in dicots. Additionally, the pDC45_dsg construct, combining a 35Spro-tRNA_sgRNA-EU unit and PCE8pro-controlled Cas9, can achieve more than 80.0% ho/bi mutations at target sites in allotetraploid tobacco. We construct pDC45_Fast system that can simultaneously fulfill gene editing and shorten the life span of T0 generation tobacco and tomato. This study provides new tools for improving targeted gene mutagenesis in dicots, and makes manipulations of genes in Solanum more feasible.

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Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Viruses ◽

10.3390/v13050779 ◽

2021 ◽

Vol 13 (5) ◽

pp. 779

Author(s):

Man Teng ◽

Yongxiu Yao ◽

Venugopal Nair ◽

Jun Luo

Keyword(s):

Biological Sciences ◽

Gene Therapy ◽

Genetic Engineering ◽

Gene Function ◽

Viral Genome ◽

Gene Editing ◽

Vaccine Development ◽

Future Prospects ◽

Dna Viruses ◽

Viral Genomes

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

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Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

Journal of Fungi ◽

10.3390/jof7070505 ◽

2021 ◽

Vol 7 (7) ◽

pp. 505

Author(s):

Ping Zhang ◽

Yu Wang ◽

Chenxi Li ◽

Xiaoyu Ma ◽

Lan Ma ◽

...

Keyword(s):

Genome Editing ◽

Fungal Pathogens ◽

Gene Editing ◽

Recombination Frequency ◽

Target Sequence ◽

Widespread Application ◽

Genetic Studies ◽

Efficient Gene ◽

Cryptococcus Species ◽

Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

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Toward a Better Understanding of Metal Nanoparticles, a Novel Strategy from Eucalyptus Plants

Plants ◽

10.3390/plants10050929 ◽

2021 ◽

Vol 10 (5) ◽

pp. 929

Author(s):

Hanadi Sawalha ◽

Rambod Abiri ◽

Ruzana Sanusi ◽

Noor Azmi Shaharuddin ◽

Aida Atiqah Mohd Noor ◽

...

Keyword(s):

Metal Nanoparticles ◽

Woody Plants ◽

Woody Species ◽

Forest Products ◽

Ideal Candidate ◽

Promising Tool ◽

Novel Strategy ◽

The Ideal

Nanotechnology is a promising tool that has opened the doors of improvement to the quality of human’s lives through its potential in numerous technological aspects. Green chemistry of nanoscale materials (1–100 nm) is as an effective and sustainable strategy to manufacture homogeneous nanoparticles (NPs) with unique properties, thus making the synthesis of green NPs, especially metal nanoparticles (MNPs), the scientist’s core theme. Researchers have tested different organisms to manufacture MNPs and the results of experiments confirmed that plants tend to be the ideal candidate amongst all entities and are suitable to synthesize a wide variety of MNPs. Natural and cultivated Eucalyptus forests are among woody plants used for landscape beautification and as forest products. The present review has been written to reflect the efficacious role of Eucalyptus in the synthesis of MNPs. To better understand this, the route of extracting MNPs from plants, in general, and Eucalyptus, in particular, are discussed. Furthermore, the crucial factors influencing the process of MNP synthesis from Eucalyptus as well as their characterization and recent applications are highlighted. Information gathered in this review is useful to build a basis for new prospective research ideas on how to exploit this woody species in the production of MNPs. Nevertheless, there is a necessity to feed the scientific field with further investigations on wider applications of Eucalyptus-derived MNPs.

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In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

Genome Medicine ◽

10.1186/s13073-021-00876-0 ◽

2021 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Menglong Chen ◽

Hui Shi ◽

Shixue Gou ◽

Xiaomin Wang ◽

Lei Li ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Genome Editing ◽

Large Scale ◽

Gene Editing ◽

High Efficiency ◽

Muscle Fibers ◽

Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

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Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

International Journal of Molecular Sciences ◽

10.3390/ijms20153623 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3623 ◽

Cited By ~ 5

Author(s):

Tobias Bruegmann ◽

Khira Deecke ◽

Matthias Fladung

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Constitutive Expression ◽

Gc Content ◽

Seed Region ◽

Research Topics ◽

Target Region ◽

Cas9 Nuclease ◽

Different Types ◽

Poplar Hybrid

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

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Alien and native woody plants in scattered vegetation in agricultural landscape

Folia Oecologica ◽

10.2478/foecol-2020-0013 ◽

2020 ◽

Vol 47 (2) ◽

pp. 109-120

Author(s):

Ján Supuka ◽

Attila Tóth ◽

Mária Bihuňová ◽

Martina Verešová ◽

Karol Šinka

Keyword(s):

Tree Species ◽

Woody Plants ◽

Agricultural Landscape ◽

Woody Plant ◽

Robinia Pseudoacacia ◽

Woody Species ◽

Fruit Trees ◽

Lycium Barbarum ◽

Dominance Index ◽

Acer Campestre

AbstractThe woody plant species composition has been evaluated in three cadastral territories of southwestern Slovakia, together in 77 habitats of non-forest woody vegetation (NFWV). A total of 43 tree species have been identified; 8 of them were alien and 5 species were cultural fruit trees. In total 20 shrub species were identified, out of which 3 were alien. Three woody species are classified as invasive according to the law in Slovakia: Acer negundo L., Ailanthus altissima (Mill.) Swingle, and Lycium barbarum L. They occurred only in 2, maximum in 4 of the evaluated habitats. The most occurring alien tree species Robinia pseudoacacia L. was generally identified in 58 habitats and in 48 habitats, with an incidence over 40% and dominance index of 70.6. The second most occurring alien tree Populus × canadensis had a dominance index of 8.3. The dominant native trees in NFWV were Acer campestre L., Fraxinus excelsior L., Quercus robur L., Salix fragilis L. with the dominance index of 1–5 only.

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Highly efficient generation of canker resistant sweet orange enabled by an improved CRISPR/Cas9 system

10.1101/2021.10.20.465103 ◽

2021 ◽

Author(s):

Xiaoen Huang ◽

Nian Wang

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Target Genes ◽

High Efficiency ◽

Sweet Orange ◽

Susceptibility Gene ◽

Transformation Rate ◽

Biallelic Mutation ◽

Important Species ◽

Citrus Production

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously (dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, CsU6 to drive sgRNA expression), we further improved our CRISPR/Cas9 system by choosing superior promoters (CmYLCV or CsUbi promoter) to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

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