scholarly journals The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants

2021 ◽  
Vol 12 ◽  
Author(s):  
Marta Vazquez-Vilar ◽  
Víctor Garcia-Carpintero ◽  
Sara Selma ◽  
Joan M. Bernabé-Orts ◽  
Javier Sanchez-Vicente ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Repetitive Sequences ◽  
Plant Genome ◽  
Luciferase Reporter ◽  
Stress Tests ◽  
Web Based ◽  
Open Platform ◽  
Guide Rnas ◽  
As Stress ◽  
Biallelic Mutations

CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, the design and assembly of multiplex constructs comprising tandemly arrayed guide RNAs (gRNAs) requires scarless cloning and is still troublesome due to the presence of repetitive sequences, thus hampering a more widespread use. Here we present a comprehensive extension of the software-assisted cloning platform GoldenBraid (GB), in which, on top of its multigene cloning software, we integrate new tools for the Type IIS-based easy and rapid assembly of up to six tandemly-arrayed gRNAs with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the crRNA unspaced approaches, respectively. As stress tests for the new tools, we assembled and used for Agrobacterium-mediated stable transformation a 17 Cas9-gRNAs construct targeting a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in Nicotiana tabacum. The 14 selected genes are targets of miR156, thus potentially playing an important role in juvenile-to-adult and vegetative-to-reproductive phase transitions. With the 17 gRNAs construct we generated a collection of Cas9-free SPL edited T1 plants harboring up to 9 biallelic mutations and showing leaf juvenility and more branching. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR/Cas activators and repressors using single and multiplexing gRNAs was validated using a Luciferase reporter with the Solanum lycopersicum Mtb promoter or the Agrobacterium tumefaciens nopaline synthase promoter in transient expression in Nicotiana benthamiana. With the incorporation of the new web-based tools and the accompanying collection of DNA parts, the GB4.0 genome edition turns an all-in-one open platform for plant genome engineering.

scholarly journals Edition of complex gene families in tobacco with GoldenBraid 4.0, a multipurpose web-based platform for plant genome engineering

2020 ◽  
Author(s):  
Marta Vazquez-Vilar ◽  
Víctor Garcia-Carpintero ◽  
Sara Selma ◽  
Joan M Bernabé-Orts ◽  
Javier Sanchez-Vicente ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Gene Families ◽  
Two Dimensions ◽  
Plant Genome ◽  
Web Based ◽  
Open Platform ◽  
Squamosa Promoter Binding Protein ◽  
Biallelic Mutations ◽  
Polyploid Crops ◽  
Game Changer

ABSTRACTCRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy in polyploid crops. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, multiplex constructs comprising tandemly arrayed gRNAs are difficult to assemble, this hampering more widespread use. Here we present a comprehensive upgrade of the popular cloning platform GoldenBraid (GB), in which, on top of its classical multigene cloning software, we integrate new assembly tools for two-dimensions gRNA multiplexing with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the gRNA unspaced approaches, respectively. As functional validation, we show, among others, the assembly of up to 17 tandemly-arrayed gRNAs constructs against a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in tobacco. With these constructs we generated a collection of Cas9-free SPL mutants harboring up to 9 biallelic mutations in a single generation. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR activators and repressors using single and multiplexing gRNAs is also validated. With the incorporation of the new CRISPR tools and part’s collection, GB4.0 turns an unprecedentedly comprehensive open platform for plant genetic engineering.

scholarly journals DNA Replicons for Plant Genome Engineering

2014 ◽  
Vol 26 (1) ◽  
pp. 151-163 ◽  
Author(s):  
Nicholas J. Baltes ◽  
Javier Gil-Humanes ◽  
Tomas Cermak ◽  
Paul A. Atkins ◽  
Daniel F. Voytas
Keyword(s):  
Genome Engineering ◽  
Plant Genome

scholarly journals Impact of CRISPR-Cas9-Based Genome Engineering in Farm Animals

2021 ◽  
Vol 8 (7) ◽  
pp. 122
Author(s):  
Parul Singh ◽  
Syed Azmal Ali
Keyword(s):  
Disease Resistance ◽  
Genome Engineering ◽  
Animal Health ◽  
Fundamental Principle ◽  
Farm Animals ◽  
Web Based ◽  
Knock Out ◽  
Guide Rna ◽  
Female Birth ◽  
Comprehensive Information

Humans are sorely over-dependent on livestock for their daily basic need of food in the form of meat, milk, and eggs. Therefore, genetic engineering and transgenesis provide the opportunity for more significant gains and production in a short span of time. One of the best strategies is the genetic alteration of livestock to enhance the efficiency of food production (e.g., meat and milk), animal health, and welfare (animal population and disease). Moreover, genome engineering in the bovine is majorly focused on subjects such as disease resistance (e.g., tuberculosis), eradicate allergens (e.g., beta-lactoglobulin knock-out), products generation (e.g., meat from male and milk from female), male or female birth specifically (animal sexing), the introduction of valuable traits (e.g., stress tolerance and disease resistance) and their wellbeing (e.g., hornlessness). This review addressed the impressive genome engineering method CRISPR, its fundamental principle for generating highly efficient target-specific guide RNA, and the accompanying web-based tools. However, we have covered the remarkable roadmap of the CRISPR method from its conception to its use in cattle. Additionally, we have updated the comprehensive information on CRISPR-based gene editing in cattle.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Hyeonjin Kim ◽  
Bora Lee ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning annealed products of two oligonucleotides harboring target-binding sequence between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

2020 ◽  
Author(s):  
Anindya Bandyopadhyay ◽  
Nagesh Kancharla ◽  
vivek javalkote ◽  
santanu dasgupta ◽  
Thomas Brutnell
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Temperature Stability ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Strand Breaks ◽  
Guide Rna ◽  
Versatile Tool ◽  
Type V ◽  
Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

scholarly journals Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish

Biology ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 102
Author(s):  
De-Li Shi
Keyword(s):  
Germ Line ◽  
Gene Products ◽  
Transgenic Expression ◽  
Guide Rna ◽  
Guide Rnas ◽  
Transgenic Embryos ◽  
Mutant Phenotypes ◽  
Biallelic Mutations ◽  
Genome Activation ◽  
Maternal Gene

Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.

scholarly journals An enormous Paris polyphylla genome sheds light on genome size evolution and polyphyllin biogenesis

2020 ◽  
Author(s):  
Jing Li ◽  
Meiqi Lv ◽  
Lei Du ◽  
A Yunga ◽  
Shijie Hao ◽  
...  
Keyword(s):  
Genome Size ◽  
Single Molecule ◽  
Repetitive Sequences ◽  
Plant Genome ◽  
Growth Stages ◽  
Evolutionary Trend ◽  
Rna Seq ◽  
Genome Size Evolution ◽  
Size Evolution ◽  
Paris Polyphylla

AbstractThe monocot family Melanthiaceae with varying genome sizes in a range of 230-fold is an ideal model to study the genome size fluctuation in plants. Its family member Paris genus demonstrates an evolutionary trend of bearing huge genomes characterized by an average c-value of 49.22 pg. Here, we report a 70.18 Gb genome assembly out of the 82.55 Gb genome of Paris polyphylla var. yunnanensis (PPY), which represents the biggest sequenced genome to date. We annotate 69.53% repetitive sequences in this genome and 62.50% of which are long-terminal repeat (LTR) transposable elements. Further evolution analysis indicates that the giant genome likely results from the joint effect of common and species-specific expansion of different LTR superfamilies, which might contribute to the environment adaptation after speciation. Moreover, we identify the candidate pathway genes for the biogenesis of polyphyllins, the PPY-specific medicinal saponins, by complementary approaches including genome mining, comprehensive analysis of 31 next-generation RNA-seq data and 55.23 Gb single-molecule circular consensus sequencing (CCS) RNA-seq reads, and correlation of the transcriptome and phytochemical data of five different tissues at four growth stages. This study not only provides significant insights into plant genome size evolution, but also paves the way for the following polyphyllin synthetic biology.

scholarly journals Electromagnetic Emissions from Quartz Subjected to Shear Stress: Spectral Signatures and Geophysical Implications

Geosciences ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 140 ◽  
Author(s):  
Giovanni Martinelli ◽  
Paolo Plescia ◽  
Emanuela Tempesta
Keyword(s):  
Shear Stress ◽  
Electromagnetic Waves ◽  
Emission Spectra ◽  
Stress Tests ◽  
Quartz Crystals ◽  
Piston Cylinder ◽  
Fracturing Process ◽  
Electromagnetic Emissions ◽  
As Stress ◽  
Energy Dissipated

Shear tests on quartz rocks and single quartz crystals have been conducted to understand the possible relationship between the intensity of detectable stress in fault areas and the energy released in the form of electromagnetic waves in the range 30 KHz-1 MHz (LF–MF). For these tests, a new type of piston-cylinder has been developed, instrumented to collect the electromagnetic signals generated by the quartz during shear stress tests and that allows energy measurements on electromagnetic emissions (EMR) to be performed. The data obtained indicate that shear-stressed quartz crystals can generate electromagnetic emissions in the LF–MF range. These emissions represent a tiny fraction of the total energy dissipated in the fracturing process. The spectrum of radio emissions consists of continuous radiation and overlapping peaks. For the first time, a characteristic migration of peak frequencies was observed, proportional to the evolution of the fracturing process. In particular, the continuous recording of the radio emission spectra shows a migration of the peaks toward higher frequencies, as stress continues over time and smaller and larger fractures form. This migration could be used to distinguish possible natural signals emitted by quartz in tectonically active environments from possible signals of other geophysical and possibly anthropogenic origin.

Recent advances in DNA-free editing and precise base editing in plants

2017 ◽  
Vol 1 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Yi Zhang ◽  
Caixia Gao
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Plant Genome ◽  
The Novel ◽  
Future Perspectives ◽  
Delivery Methods ◽  
Base Editing ◽  
Short Palindromic Repeat ◽  
Target Effects

Genome-editing technologies based on the CRISPR (clustered regularly interspaced short palindromic repeat) system have been widely used in plants to investigate gene function and improve crop traits. The recently developed DNA-free delivery methods and precise base-editing systems provide new opportunities for plant genome engineering. In this review, we describe the novel DNA-free genome-editing methods in plants. These methods reduce off-target effects and may alleviate regulatory concern about genetically modified plants. We also review applications of base-editing systems, which are highly effective in generating point mutations and are of great value for introducing agronomically valuable traits. Future perspectives for DNA-free editing and base editing are also discussed.

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