scholarly journals A potential clue for the application of prime editing in tomato, a dicot plant

2021 ◽  
Author(s):  
Tien Van Vu ◽  
Jihae Kim ◽  
Swati Das ◽  
Jae-Yean Kim
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Crop Improvement ◽  
Plant Genome ◽  
Guide Rna ◽  
Genome Modification ◽  
Potential Applications ◽  
Targeted Deep Sequencing ◽  
Synthetic Sequence

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a priming extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. However, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. However, our data obtained by targeted deep sequencing also revealed inefficient PE activity in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. These data could also help explain the recent successes of some prime editors in plants using improved expression systems. Our work provides an important clue for the application of the PE approach in crop improvement.

scholarly journals Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Wen Xu ◽  
Wei Song ◽  
Yongxing Yang ◽  
Ying Wu ◽  
Xinxin Lv ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Crop Improvement ◽  
High Fidelity ◽  
Knock Out ◽  
Base Editing ◽  
Guide Rna ◽  
Genome Modification ◽  
Targeted Genome Modification ◽  
Target Effect

Abstract Background Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. Results We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture. Conclusions We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.

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 Efficient targeted integration directed by short homology in zebrafish and mammalian cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Wesley A Wierson ◽  
Jordan M Welker ◽  
Maira P Almeida ◽  
Carla M Mann ◽  
Dennis A Webster ◽  
...  
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
High Efficiency ◽  
Cost Effective ◽  
Strand Break ◽  
End Joining ◽  
Cost Effective Approach ◽  
Targeted Integration

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

scholarly journals Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice

Genes ◽  
2019 ◽  
Vol 10 (8) ◽  
pp. 596 ◽  
Author(s):  
Haroon Butt ◽  
Agnieszka Piatek ◽  
Lixin Li ◽  
Anireddy S. N. Reddy ◽  
Magdy M. Mahfouz
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Stress Responses ◽  
Genome Engineering ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Plant Genome ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
A Genome

Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses.

scholarly journals Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox

2019 ◽  
Vol 20 (16) ◽  
pp. 4045 ◽  
Author(s):  
Ali Razzaq ◽  
Fozia Saleem ◽  
Mehak Kanwal ◽  
Ghulam Mustafa ◽  
Sumaira Yousaf ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Crop Improvement ◽  
Crop Plants ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
Zinc Finger Nucleases ◽  
Base Substitution ◽  
Transcription Activator ◽  
Abiotic Stressors

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

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

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Expression System ◽  
Plant Genome ◽  
Rna Expression ◽  
Guide Rna

scholarly journals SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Ziyan Xu ◽  
Yongjie Kuang ◽  
Bin Ren ◽  
Daqi Yan ◽  
Fang Yan ◽  
...  
Keyword(s):  
Transgenic Rice ◽  
High Frequency ◽  
Genome Engineering ◽  
Target Genes ◽  
Cytidine Deaminase ◽  
Nuclease Activity ◽  
The Self ◽  
Plant Genome ◽  
Dna Fragments ◽  
Wide Range

Abstract Background Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. Results In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. Conclusions The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.

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

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
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 Background: The 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.Results: We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand 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.Conclusions: This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals CRISPR/dCas9-based Systems: Mechanisms and Applications in Plant Sciences

2021 ◽  
Author(s):  
Chou Khai Soong Karlson ◽  
Siti Nurfadhlina Mohd Noor ◽  
Nadja Nolte ◽  
Boon Chin Tan
Keyword(s):  
Genetically Modified Organisms ◽  
Crop Improvement ◽  
Gene Activation ◽  
Delivery Methods ◽  
Guide Rna ◽  
Efficient Delivery ◽  
Potential Applications ◽  
Transcriptional Modulation ◽  
Plant Sciences ◽  
Target Effects

RNA-guided genomic transcriptional regulation tools, namely Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) and CRISPR-mediated gene activation (CRISPRa), are a powerful technology for the field of functional genomics. Deriving from the CRISPR/Cas9 system, both systems comprise a catalytically dead Cas9 (dCas9) and a single guide RNA (sgRNA). This type of dCas9 is incapable of cleaving DNA but retains its ability to specifically bind to DNA. The binding of the dCas9/sgRNA complex to a target gene results in transcriptional interference. The CRISPR/dCas9 system has been explored as a tool for transcriptional modulation and genome imaging. Despite its potential applications and benefits, the challenges and limitations faced by the CRISPR/dCas9 system include the off-target effects, PAM sequence requirement, efficient delivery methods, and the CRISPR/dCas9-interfered crops being labeled as genetically modified organisms in several countries. This review highlights the progression of CRISPR/dCas9 technology as well as its applications and potential challenges in crop improvement.

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