scholarly journals Comparison of Efficiency and Specificity of CRISPR-Associated (Cas) Nucleases in Plants: An Expanded Toolkit for Precision Genome Engineering

2018 ◽  
Author(s):  
Oleg Raitskin ◽  
Christian Schudoma ◽  
Anthony West ◽  
Nicola J. Patron
Keyword(s):  
Plant Species ◽  
Genome Engineering ◽  
Bacterial Species ◽  
Gene Families ◽  
Plant Genome ◽  
Parental Line ◽  
High Fidelity ◽  
Related Gene ◽  
Molecular Tools ◽  
Comparative Performance

Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called ‘off-targets’ by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content correlates with efficiency and that Cas9 from Staphylococcus aureus is comparatively most efficient at inducing mutations. We also demonstrate that ‘high-fidelity’ variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.

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 Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Yi Zhang ◽  
Zhen Liang ◽  
Yuan Zong ◽  
Yanpeng Wang ◽  
Jinxing Liu ◽  
...  
Keyword(s):  
Durum Wheat ◽  
Genome Editing ◽  
Bread Wheat ◽  
Transient Expression ◽  
Plant Species ◽  
Genome Engineering ◽  
Plant Genome ◽  
Engineering Research ◽  
Plant Genomes ◽  
Highly Efficient

Abstract Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.

scholarly journals Phylogenetic analysis of pectin-related gene families in Physcomitrella patens and nine other plant species yields evolutionary insights into cell walls

2014 ◽  
Vol 14 (1) ◽  
pp. 79 ◽  
Author(s):  
Thomas W McCarthy ◽  
Joshua P Der ◽  
Loren A Honaas ◽  
Claude W dePamphilis ◽  
Charles T Anderson
Keyword(s):  
Phylogenetic Analysis ◽  
Plant Species ◽  
Cell Walls ◽  
Physcomitrella Patens ◽  
Gene Families ◽  
Related Gene

scholarly journals A Genomic Perspective on the Evolutionary Diversity of the Plant Cell Wall

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1195 ◽  
Author(s):  
Ryusuke Yokoyama
Keyword(s):  
Cell Wall ◽  
Plant Species ◽  
Functional Diversity ◽  
Plant Cell ◽  
Cell Walls ◽  
Plant Cell Wall ◽  
Dynamic Structure ◽  
Gene Families ◽  
Plant Genome ◽  
History Of

The plant cell wall is a complex and dynamic structure composed of numerous different molecules that play multiple roles in all aspects of plant life. Currently, a new frontier in biotechnology is opening up, which is providing new insights into the structural and functional diversity of cell walls, and is thus serving to re-emphasize the significance of cell wall divergence in the evolutionary history of plant species. The ever-increasing availability of plant genome datasets will thus provide an invaluable basis for enhancing our knowledge regarding the diversity of cell walls among different plant species. In this review, as an example of a comparative genomics approach, I examine the diverse patterns of cell wall gene families among 100 species of green plants, and illustrate the evident benefits of using genome databases for studying cell wall divergence. Given that the growth and development of all types of plant cells are intimately associated with cell wall dynamics, gaining a further understanding of the functional diversity of cell walls in relation to diverse biological events will make significant contributions to a broad range of plant sciences.

scholarly journals CRISPR-based tools for plant genome engineering

2017 ◽  
Vol 1 (2) ◽  
pp. 135-149 ◽  
Author(s):  
Nathalia Volpi e Silva ◽  
Nicola J. Patron
Keyword(s):  
Genome Engineering ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
Molecular Tools ◽  
Cas9 Protein ◽  
Wide Range ◽  
Short Palindromic Repeat ◽  
Modulation Of Gene Expression ◽  
Multiple Sequences ◽  
Rna Complexes

Molecular tools adapted from bacterial CRISPR (clustered regulatory interspaced short palindromic repeat) adaptive immune systems have been demonstrated in an increasingly wide range of plant species. They have been applied for the induction of targeted mutations in one or more genes as well as for directing the integration of new DNA to specific genomic loci. The construction of molecular tools for multiplexed CRISPR-mediated editing in plants has been facilitated by cloning techniques that allow multiple sequences to be assembled together in a single cloning reaction. Modifications of the canonical Cas9 protein from Streptococcus pyogenes and the use of nucleases from other bacteria have increased the diversity of genomic sequences that can be targeted and allow the delivery of protein cargos such as transcriptional activators and repressors. Furthermore, the direct delivery of protein–RNA complexes to plant cells and tissues has enabled the production of engineered plants without the delivery or genomic integration of foreign DNA. Here, we review toolkits derived from bacterial CRISPR systems for targeted mutagenesis, gene delivery and modulation of gene expression in plants, focusing on their composition and the strategies employed to reprogramme them for the recognition of specific genomic targets.

scholarly journals Patient-Specific Predictive Antibiogram in Decision Support for Empiric Antibiotic Treatment

2020 ◽  
Vol 41 (S1) ◽  
pp. s521-s522
Author(s):  
Debarka Sengupta ◽  
Vaibhav Singh ◽  
Seema Singh ◽  
Dinesh Tewari ◽  
Mudit Kapoor ◽  
...  
Keyword(s):  
Machine Learning ◽  
Antimicrobial Resistance ◽  
Model Building ◽  
Medical Center ◽  
Bacterial Species ◽  
Model Performance ◽  
The United States ◽  
Patient Specific ◽  
Gradient Boosting ◽  
Comparative Performance

Background: The rising trend of antibiotic resistance imposes a heavy burden on healthcare both clinically and economically (US$55 billion), with 23,000 estimated annual deaths in the United States as well as increased length of stay and morbidity. Machine-learning–based methods have, of late, been used for leveraging patient’s clinical history and demographic information to predict antimicrobial resistance. We developed a machine-learning model ensemble that maximizes the accuracy of such a drug-sensitivity versus resistivity classification system compared to the existing best-practice methods. Methods: We first performed a comprehensive analysis of the association between infecting bacterial species and patient factors, including patient demographics, comorbidities, and certain healthcare-specific features. We leveraged the predictable nature of these complex associations to infer patient-specific antibiotic sensitivities. Various base-learners, including k-NN (k-nearest neighbors) and gradient boosting machine (GBM), were used to train an ensemble model for confident prediction of antimicrobial susceptibilities. Base learner selection and model performance evaluation was performed carefully using a variety of standard metrics, namely accuracy, precision, recall, F1 score, and Cohen κ. Results: For validating the performance on MIMIC-III database harboring deidentified clinical data of 53,423 distinct patient admissions between 2001 and 2012, in the intensive care units (ICUs) of the Beth Israel Deaconess Medical Center in Boston, Massachusetts. From ~11,000 positive cultures, we used 4 major specimen types namely urine, sputum, blood, and pus swab for evaluation of the model performance. Figure 1 shows the receiver operating characteristic (ROC) curves obtained for bloodstream infection cases upon model building and prediction on 70:30 split of the data. We received area under the curve (AUC) values of 0.88, 0.92, 0.92, and 0.94 for urine, sputum, blood, and pus swab samples, respectively. Figure 2 shows the comparative performance of our proposed method as well as some off-the-shelf classification algorithms. Conclusions: Highly accurate, patient-specific predictive antibiogram (PSPA) data can aid clinicians significantly in antibiotic recommendation in ICU, thereby accelerating patient recovery and curbing antimicrobial resistance.Funding: This study was supported by Circle of Life Healthcare Pvt. Ltd.Disclosures: None

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 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 Spontaneous adaptation of ion selectivity in a bacterial flagellar motor

2021 ◽  
Author(s):  
Pietro Ridone ◽  
Tsubasa Ishida ◽  
Yoshiyuki Sowa ◽  
Matthew A. B. Baker
Keyword(s):  
Genome Engineering ◽  
Environmental Changes ◽  
Ion Selectivity ◽  
Bacterial Species ◽  
Selective Advantage ◽  
Flagellar Motor ◽  
Novel Mutations ◽  
Power Sources ◽  
Motor Complex ◽  
Bacterial Flagellar Motor

ABSTRACTMotility provides a selective advantage to many bacterial species and is often achieved by rotation of flagella that propel the cell towards more favourable conditions. In most species, the rotation of the flagellum, driven by the Bacterial Flagellar Motor (BFM), is powered by H+ or Na+ ion transit through the torque-generating stator subunits of the motor complex. The ionic requirements for motility appear to have adapted to environmental changes throughout history but the molecular basis of this adaptation, and the constraints which govern the evolution of the stator proteins are unknown. Here we use CRISPR-mediated genome engineering to replace the native H+-powered stator genes of Escherichia coli with a compatible sodium-powered stator set from Vibrio alginolyticus and subsequently direct the evolution of the stators to revert to H+-powered motility. Evidence from whole genome sequencing indicates both flagellar- and non-flagellar-associated genes that are involved in longer-term adaptation to new power sources. Overall, transplanted Na+-powered stator genes can spontaneously incorporate novel mutations that allow H+-motility when environmental Na+ is lacking.

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