Precision Genome Engineering Through Cytidine Base Editing in Rapeseed (Brassica napus. L)

Rapeseed is one of the world's most important sources of oilseed crops. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Therefore, genome-wide and target-specific base editing will greatly facilitate precision plant molecular breeding. In this study, four CBE systems (BnPBE, BnA3A-PBE, BnA3A1-PBE, and BnPBGE14) were modified to achieve cytidine base editing at five target genes in rapeseed. The results indicated that genome editing is achievable in three CBEs systems, among which BnA3A1-PBE had the highest base-editing efficiency (average 29.8% and up to 50.5%) compared to all previous CBEs reported in rapeseed. The editing efficiency of BnA3A1-PBE is ~8.0% and fourfold higher, than those of BnA3A-PBE (averaging 27.6%) and BnPBE (averaging 6.5%), respectively. Moreover, BnA3A1-PBE and BnA3A-PBE could significantly increase the proportion of both the homozygous and biallelic genotypes, and also broaden the editing window compared to BnPBE. The cytidine substitution which occurred at the target sites of both BnaA06.RGA and BnaALS were stably inherited and conferred expected gain-of-function phenotype in the T1 generation (i.e., dwarf phenotype or herbicide resistance for weed control, respectively). Moreover, new alleles or epialleles with expected phenotype were also produced, which served as an important resource for crop improvement. Thus, the improved CBE system in the present study, BnA3A1-PBE, represents a powerful base editor for both gene function studies and molecular breeding in rapeseed.

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Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

aBIOTECH ◽

10.1007/s42994-021-00042-5 ◽

2021 ◽

Author(s):

Jun Li ◽

Yan Li ◽

Ligeng Ma

Keyword(s):

Functional Genomics ◽

Genome Editing ◽

Functional Annotation ◽

Genome Engineering ◽

Agronomic Traits ◽

Wheat Breeding ◽

Breeding Programs ◽

Base Editing ◽

The World ◽

World Food Security

AbstractCommon wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

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Genome Editing in Cereals: Approaches, Applications and Challenges

International Journal of Molecular Sciences ◽

10.3390/ijms21114040 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4040 ◽

Cited By ~ 4

Author(s):

Waquar A. Ansari ◽

Sonali U. Chandanshive ◽

Vacha Bhatt ◽

Altafhusain B. Nadaf ◽

Sanskriti Vats ◽

...

Keyword(s):

Genome Editing ◽

Target Genes ◽

Crop Improvement ◽

Whole Genome Sequence ◽

Cereal Crops ◽

Sequence Information ◽

World Population ◽

Base Editing ◽

Crop Varieties ◽

Current Scenario

Over the past decades, numerous efforts were made towards the improvement of cereal crops mostly employing traditional or molecular breeding approaches. The current scenario made it possible to efficiently explore molecular understanding by targeting different genes to achieve desirable plants. To provide guaranteed food security for the rising world population particularly under vulnerable climatic condition, development of high yielding stress tolerant crops is needed. In this regard, technologies upgradation in the field of genome editing looks promising. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a rapidly growing genome editing technique being effectively applied in different organisms, that includes both model and crop plants. In recent times CRISPR/Cas9 is being considered as a technology which revolutionized fundamental as well as applied research in plant breeding. Genome editing using CRISPR/Cas9 system has been successfully demonstrated in many cereal crops including rice, wheat, maize, and barley. Availability of whole genome sequence information for number of crops along with the advancement in genome-editing techniques provides several possibilities to achieve desirable traits. In this review, the options available for crop improvement by implementing CRISPR/Cas9 based genome-editing techniques with special emphasis on cereal crops have been summarized. Recent advances providing opportunities to simultaneously edit many target genes were also discussed. The review also addressed recent advancements enabling precise base editing and gene expression modifications. In addition, the article also highlighted limitations such as transformation efficiency, specific promoters and most importantly the ethical and regulatory issues related to commercial release of novel crop varieties developed through genome editing.

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A Cas9 with Complete PAM Recognition for Adenine Dinucleotides

10.1101/429654 ◽

2018 ◽

Cited By ~ 8

Author(s):

Noah Jakimo ◽

Pranam Chatterjee ◽

Lisa Nip ◽

Joseph M Jacobson

Keyword(s):

Streptococcus Pyogenes ◽

Sequence Space ◽

Genome Engineering ◽

Protein Domains ◽

Human Cells ◽

Gene Modification ◽

Editing Efficiency ◽

Base Editing ◽

Protospacer Adjacent Motif

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a Type-IIA CRISPR-associated enzyme through identification of the natural 5’-NAA-3’ PAM specificity of a Streptococcus macacae Cas9 (Smac Cas9). We further recombine protein domains between Smac Cas9 and its well-established ortholog from Streptococcus pyogenes (Spy Cas9), as well as an “increased” nucleolytic variant (iSpy Cas9), to achieve consistent mediation of gene modification and base editing. In a comparison to previously reported Cas9 and Cas12a enzymes, we show that our hybrids recognize all adenine dinucleotide PAM sequences and possess robust editing efficiency in human cells.

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Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

Nature Communications ◽

10.1038/s41467-020-18715-y ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Minh Thuan Nguyen Tran ◽

Mohd Khairul Nizam Mohd Khalid ◽

Qi Wang ◽

Jacqueline K. R. Walker ◽

Grace E. Lidgerwood ◽

...

Keyword(s):

Genome Engineering ◽

Editing Efficiency ◽

Base Editing ◽

Target Dna ◽

Dna Editing ◽

Target Activity ◽

Adenine Base

Abstract Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

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Rolling Circle Amplification (RCA)-Mediated Genome-Wide ihpRNAi Mutant Library Construction in Brassica napus

International Journal of Molecular Sciences ◽

10.3390/ijms21197243 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7243

Author(s):

Shengbo Zhao ◽

Junling Luo ◽

Xinhua Zeng ◽

Keqi Li ◽

Rong Yuan ◽

...

Keyword(s):

Brassica Napus ◽

Functional Genomics ◽

Insertional Mutagenesis ◽

Molecular Mechanisms ◽

Target Genes ◽

Agronomic Traits ◽

Rolling Circle Amplification ◽

Functional Characterization ◽

Rolling Circle ◽

Genome Wide

With the successful completion of genomic sequencing for Brassica napus, identification of novel genes, determination of functions performed by genes, and exploring the molecular mechanisms underlying important agronomic traits were challenged. Mutagenesis-based functional genomics techniques including chemical, physical, and insertional mutagenesis have been used successfully in the functional characterization of genes. However, these techniques had their disadvantages and inherent limitations for allopolyploid Brassica napus, which contained a large number of homologous and redundant genes. Long intron-spliced hairpin RNA (ihpRNA) constructs which contained inverted repeats of the target gene separated by an intron, had been shown to be very effective in triggering RNAi in plants. In the present study, the genome-wide long ihpRNA library of B. napus was constructed with the rolling circle amplification (RCA)-mediated technology. Using the phytoene desaturase (PDS) gene as a target control, it was shown that the RCA-mediated long ihpRNA construct was significantly effective in triggering gene silence in B. napus. Subsequently, the resultant long ihpRNA library was transformed into B. napus to produce corresponding RNAi mutants. Among the obtained transgenic ihpRNA population of B. napus, five ihpRNA lines with observable mutant phenotypes were acquired including alterations in the floral model and the stamen development. The target genes could be quickly identified using specific primers. These results showed that the RCA-mediated ihpRNA construction method was effective for the genome-wide long ihpRNA library of B. napus, therefore providing a platform for study of functional genomics in allopolyploid B. napus.

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CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.

International Journal of Molecular Sciences ◽

10.3390/ijms19092716 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2716 ◽

Cited By ~ 19

Author(s):

Qinfu Sun ◽

Li Lin ◽

Dongxiao Liu ◽

Dewei Wu ◽

Yujie Fang ◽

...

Keyword(s):

Brassica Napus ◽

Transgenic Plants ◽

Genome Editing ◽

Target Genes ◽

High Efficiency ◽

Pathogenic Fungus ◽

Crop Improvement ◽

Brassica Napus L ◽

Significant Difference ◽

Genomic Editing

Targeted genome editing is a desirable means of basic science and crop improvement. The clustered, regularly interspaced, palindromic repeat (CRISPR)/Cas9 (CRISPR-associated 9) system is currently the simplest and most commonly used system in targeted genomic editing in plants. Single and multiplex genome editing in plants can be achieved under this system. In Arabidopsis, AtWRKY11 and AtWRKY70 genes were involved in JA- and SA-induced resistance to pathogens, in rapeseed (Brassica napus L.), BnWRKY11 and BnWRKY70 genes were found to be differently expressed after inoculated with the pathogenic fungus, Sclerotinia sclerotiorum (Lib.) de Bary. In this study, two Cas9/sgRNA constructs targeting two copies of BnWRKY11 and four copies of BnWRKY70 were designed to generate BnWRKY11 and BnWRKY70 mutants respectively. As a result, twenty-two BnWRKY11 and eight BnWRKY70 independent transformants (T0) were obtained, with the mutation ratios of 54.5% (12/22) and 50% (4/8) in BnWRKY11 and BnWRKY70 transformants respectively. Eight and two plants with two copies of mutated BnWRKY11 and BnWRKY70 were obtained respectively. In T1 generation of each plant examined, new mutations on target genes were detected with high efficiency. The vast majority of BnWRKY70 mutants showed editing in three copies of BnWRKY70 in examined T1 plants. BnWRKY70 mutants exhibited enhanced resistance to Sclerotinia, while BnWRKY11 mutants showed no significant difference in Sclerotinia resistance when compared to non-transgenic plants. In addition, plants that overexpressed BnWRKY70 showed increased sensitivity when compared to non-transgenic plants. Altogether, our results demonstrated that BnWRKY70 may function as a regulating factor to negatively control the Sclerotinia resistance and CRISPR/Cas9 system could be used to generate germplasm in B. napus with high resistance against Sclerotinia.

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citSATdb: Genome-Wide Simple Sequence Repeat (SSR) Marker Database of Citrus Species for Germplasm Characterization and Crop Improvement

Genes ◽

10.3390/genes11121486 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1486

Author(s):

Naveen Duhan ◽

Manish Meshram ◽

Cristian D. Loaiza ◽

Rakesh Kaundal

Keyword(s):

Ssr Markers ◽

Molecular Breeding ◽

Crop Improvement ◽

Variety Identification ◽

Germplasm Characterization ◽

Citrus Species ◽

Genome Wide ◽

Genomic Regions ◽

Chromosome Scaffold ◽

Simple Sequence

Microsatellites or simple sequence repeats (SSRs) are popular co-dominant markers that play an important role in crop improvement. To enhance genomic resources in general horticulture, we identified SSRs in the genomes of eight citrus species and characterized their frequency and distribution in different genomic regions. Citrus is the world’s most widely cultivated fruit crop. We have implemented a microsatellite database, citSATdb, having the highest number (~1,296,500) of putative SSR markers from the genus Citrus, represented by eight species. The database is based on a three-tier approach using MySQL, PHP, and Apache. The markers can be searched using multiple search parameters including chromosome/scaffold number(s), motif types, repeat nucleotides (1–6), SSR length, patterns of repeat motifs and chromosome/scaffold location. The cross-species transferability of selected markers can be checked using e-PCR. Further, the markers can be visualized using the Jbrowse feature. These markers can be used for distinctness, uniformity, and stability (DUS) tests of variety identification, marker-assisted selection (MAS), gene discovery, QTL mapping, and germplasm characterization. citSATdb represents a comprehensive source of markers for developing/implementing new approaches for molecular breeding, required to enhance Citrus productivity. The potential polymorphic SSR markers identified by cross-species transferability could be used for genetic diversity and population distinction in other species.

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Genetic diversity and GWAS of agronomic traits using an ICARDA lentil (Lens culinaris Medik.) Reference Plus collection

Plant Genetic Resources ◽

10.1017/s147926212100006x ◽

2021 ◽

pp. 1-10

Author(s):

Karthika Rajendran ◽

Clarice J. Coyne ◽

Ping Zheng ◽

Gopesh Saha ◽

Dorrie Main ◽

...

Keyword(s):

Genetic Diversity ◽

Lens Culinaris ◽

Agronomic Traits ◽

Plant Genetic Resources ◽

Crop Improvement ◽

Principal Component ◽

Genotyping By Sequencing ◽

Phenotypic Data ◽

Reference Set ◽

Genome Wide

Abstract Genotyping of lentil plant genetic resources holds the promise to increase the identification and utilization of useful genetic diversity for crop improvement. The International Center for Agriculture Research in the Dry Areas (ICARDA) lentil reference set plus collection of 176 accessions was genotyped using genotyping-by-sequencing (GBS) and 22,555 SNPs were identified. The population structure was investigated using Bayesian analysis (STRUCTURE, k = 3) and principal component analysis. The two methods are in concordance. Genome-wide association analysis (GWAS) using the filtered SNP set and ICARDA historical phenotypic data discovered putative markers for several agronomic traits including days to first flower, seeds per pod, seed weight and days to maturity. The genetic and genomic resources developed and utilized in this study are available to the research community interested in exploring the ICARDA reference set plus collection using GWAS.

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The expanding roles of APETALA2/Ethylene Responsive Factors and their potential applications in crop improvement

Briefings in Functional Genomics ◽

10.1093/bfgp/elz001 ◽

2019 ◽

Vol 18 (4) ◽

pp. 240-254 ◽

Cited By ~ 8

Author(s):

Rajat Srivastava ◽

Rahul Kumar

Keyword(s):

Stress Responses ◽

Regulatory Networks ◽

Target Genes ◽

Agronomic Traits ◽

Crop Improvement ◽

Plant Stress ◽

Structural Features ◽

Plant Responses ◽

Ethylene Response Factor ◽

Improvement Programs

AbstractUnderstanding the molecular basis of the gene-regulatory networks underlying agronomic traits or plant responses to abiotic/biotic stresses is very important for crop improvement. In this context, transcription factors, which either singularly or in conjugation directly control the expression of many target genes, are suitable candidates for improving agronomic traits via genetic engineering. In this regard, members of one of the largest class of plant-specific APETALA2/Ethylene Response Factor (AP2/ERF) superfamily, which is implicated in various aspects of development and plant stress adaptation responses, are considered high-value targets for crop improvement. Besides their long-known regulatory roles in mediating plant responses to abiotic stresses such as drought and submergence, the novel roles of AP2/ERFs during fruit ripening or secondary metabolites production have also recently emerged. The astounding functional plasticity of AP2/ERF members is considered to be achieved by their interplay with other regulatory networks and signalling pathways. In this review, we have integrated the recently accumulated evidence from functional genomics studies and described their newly emerged functions in plants. The key structural features of AP2/ERF proteins and the modes of their action are briefly summarized. The importance of AP2/ERFs in plant development and stress responses and a summary of the event of their successful applications in crop improvement programs are also provided. Altogether, we envisage that the synthesized information presented in this review will be useful to design effective strategies for improving agronomic traits in crop plants.

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Plastid Transformation: How Does it Work? Can it Be Applied to Crops? What Can it Offer?

International Journal of Molecular Sciences ◽

10.3390/ijms21144854 ◽

2020 ◽

Vol 21 (14) ◽

pp. 4854 ◽

Cited By ~ 1

Author(s):

Yihe Yu ◽

Po-Cheng Yu ◽

Wan-Jung Chang ◽

Keke Yu ◽

Choun-Sea Lin

Keyword(s):

Chloroplast Genome ◽

Genome Engineering ◽

Agronomic Traits ◽

Crop Improvement ◽

Plastid Transformation ◽

Nuclear Genome ◽

Regeneration Ability ◽

Protein Accumulation ◽

Nuclear Transformation ◽

Alternative Means

In recent years, plant genetic engineering has advanced agriculture in terms of crop improvement, stress and disease resistance, and pharmaceutical biosynthesis. Cells from land plants and algae contain three organelles that harbor DNA: the nucleus, plastid, and mitochondria. Although the most common approach for many plant species is the introduction of foreign DNA into the nucleus (nuclear transformation) via Agrobacterium- or biolistics-mediated delivery of transgenes, plastid transformation offers an alternative means for plant transformation. Since there are many copies of the chloroplast genome in each cell, higher levels of protein accumulation can often be achieved from transgenes inserted in the chloroplast genome compared to the nuclear genome. Chloroplasts are therefore becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes. This review provides a comprehensive historical and biological perspective on plastid transformation, with a focus on current and emerging approaches such as the use of single-walled carbon nanotubes (SWNTs) as DNA delivery vehicles, overexpressing morphogenic regulators to enhance regeneration ability, applying genome editing techniques to accelerate double-stranded break formation, and reconsidering protoplasts as a viable material for plastid genome engineering, even in transformation-recalcitrant species.

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