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

aBIOTECH ◽  
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.

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

2020 ◽  
Vol 2 ◽  
Author(s):  
Limin Hu ◽  
Olalekan Amoo ◽  
Qianqian Liu ◽  
Shengli Cai ◽  
Miaoshan Zhu ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Molecular Breeding ◽  
Target Genes ◽  
Agronomic Traits ◽  
Crop Improvement ◽  
Brassica Napus L ◽  
Editing Efficiency ◽  
Base Editing ◽  
Expected Gain ◽  
Genome Wide

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.

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.

scholarly journals The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

2019 ◽  
Author(s):  
Florian Veillet ◽  
Laura Chauvin ◽  
Marie-Paule Kermarrec ◽  
François Sevestre ◽  
Mathilde Merrer ◽  
...  
Keyword(s):  
Solanum Tuberosum ◽  
Genome Editing ◽  
Genome Engineering ◽  
Basic Research ◽  
Loss Of Function ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Discrete Variation ◽  
Tetraploid Potato ◽  
Base Editing

AbstractGenome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knockout the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss-of-function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.Key MessageThe StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base editing strategies, leading to plants with impaired amylose biosynthesis.

scholarly journals Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops

2020 ◽  
Vol 21 (3) ◽  
pp. 1024 ◽  
Author(s):  
Florian Veillet ◽  
Laura Perrot ◽  
Anouchka Guyon-Debast ◽  
Marie-Paule Kermarrec ◽  
Laura Chauvin ◽  
...  
Keyword(s):  
Plant Breeding ◽  
Genome Editing ◽  
Gene Function ◽  
Physcomitrella Patens ◽  
Agronomic Traits ◽  
Function Analysis ◽  
Single Amino Acid ◽  
Base Substitution ◽  
Base Editing ◽  
Gene Function Analysis

Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.

scholarly journals Base editing and prime editing in laboratory animals

2021 ◽  
pp. 002367722199389
Author(s):  
Federico Caso ◽  
Benjamin Davies
Keyword(s):  
Genome Editing ◽  
Laboratory Animal ◽  
Genome Engineering ◽  
Point Mutations ◽  
Animal Research ◽  
Ease Of Use ◽  
Laboratory Animals ◽  
Genomic Change ◽  
Base Editing ◽  
Adenine Base

Genome editing by programmable RNA-dependent Cas endonucleases has revolutionised the field of genome engineering, achieving targeted genomic change at unprecedented efficiencies with considerable application in laboratory animal research. Despite its ease of use and wide application, there remain concerns about the precision of this technology and a number of unpredictable consequences have been reported, mostly resulting from the DNA double-strand break (DSB) that conventional CRISPR editing induces. In order to improve editing precision, several iterations of the technology been developed over the years. Base editing is one of most successful developments, allowing for single base conversions but without the need for a DSB. Cytosine and adenine base editing are now established as reliable methods to achieve precise genome editing in animal research studies. Both cytosine and adenine base editors have been applied successfully to the editing of zygotes, resulting in the generation of animal models. Similarly, both base editors have achieved precise editing of point mutations in somatic cells, facilitating the development of gene therapy approaches. Despite rapid progress in optimising these tools, base editing can address only a subset of possible base conversions within a relatively narrow window and larger genomic manipulations are not possible. The recent development of prime editing, originally defined as a simple ‘search and replace’ editing tool, may help address these limitations and could widen the range of genome manipulations possible. Preliminary reports of prime editing in animals are being published, and this new technology may allow significant advancements for laboratory animal research.

scholarly journals Plant-based biosensors for detecting CRISPR-mediated genome engineering

2021 ◽  
Author(s):  
Guoliang Yuan ◽  
Md Mahmudul Hassan ◽  
Tao Yao ◽  
Haiwei Lu ◽  
Michael Melesse Vergara ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Genome Editing ◽  
Transient Expression ◽  
Gene Editing ◽  
Genome Engineering ◽  
Protoplast Transformation ◽  
Reliable System ◽  
Base Editing ◽  
Detection Systems ◽  
Cas9 Nuclease

CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with CRISPR/Cas9 technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRIPSR/Cas tools in biological systems is therefore very necessary. Here, we developed the real-time detection systems that can spontaneously indicate CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing and CRIPSRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.

scholarly journals Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

2021 ◽  
Author(s):  
Teng-Kuei Huang ◽  
Holger Puchta
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Chromosomal Rearrangements ◽  
First Century ◽  
Chromosome Engineering ◽  
Base Editing ◽  
Cas9 Nuclease ◽  
Tremendous Progress ◽  
Genetic Linkages ◽  
Induced Mutagenesis

AbstractIn the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.

scholarly journals From Genetic Maps to QTL Cloning: An Overview for Durum Wheat

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 315
Author(s):  
Pasqualina Colasuonno ◽  
Ilaria Marcotuli ◽  
Agata Gadaleta ◽  
Jose Miguel Soriano
Keyword(s):  
Molecular Markers ◽  
Durum Wheat ◽  
Agronomic Traits ◽  
Sequence Data ◽  
Current Knowledge ◽  
Wheat Breeding ◽  
Genetic Maps ◽  
Breeding Programs ◽  
Qtl Fine Mapping ◽  
Candidate Loci

Durum wheat is one of the most important cultivated cereal crops, providing nutrients to humans and domestic animals. Durum breeding programs prioritize the improvement of its main agronomic traits; however, the majority of these traits involve complex characteristics with a quantitative inheritance (quantitative trait loci, QTL). This can be solved with the use of genetic maps, new molecular markers, phenotyping data of segregating populations, and increased accessibility to sequences from next-generation sequencing (NGS) technologies. This allows for high-density genetic maps to be developed for localizing candidate loci within a few Kb in a complex genome, such as durum wheat. Here, we review the identified QTL, fine mapping, and cloning of QTL or candidate genes involved in the main traits regarding the quality and biotic and abiotic stresses of durum wheat. The current knowledge on the used molecular markers, sequence data, and how they changed the development of genetic maps and the characterization of QTL is summarized. A deeper understanding of the trait architecture useful in accelerating durum wheat breeding programs is envisioned.

scholarly journals Enabling large-scale genome editing by reducing DNA nicking

2019 ◽  
Author(s):  
Cory J. Smith ◽  
Oscar Castanon ◽  
Khaled Said ◽  
Verena Volf ◽  
Parastoo Khoshakhlagh ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Base Editing ◽  
Single Strand Breaks ◽  
Dna Nicking ◽  
Mammalian Genomes ◽  
Induced Pluripotent

AbstractTo extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements – ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.One Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

Identification of quantitative trait loci for seedling root traits from Tibetan semi-wild wheat (Triticum aestivum subsp. tibetanum)

Genome ◽  
2017 ◽  
Vol 60 (12) ◽  
pp. 1068-1075 ◽  
Author(s):  
Jian Ma ◽  
Wei Luo ◽  
Han Zhang ◽  
Xiao-Hong Zhou ◽  
Na-Na Qin ◽  
...  
Keyword(s):  
Triticum Aestivum ◽  
Recombinant Inbred Line Population ◽  
Recombinant Inbred ◽  
Agronomic Traits ◽  
Root Traits ◽  
Wheat Breeding ◽  
Breeding Programs ◽  
Wild Wheat ◽  
Seedling Root ◽  
Variation Explained

As a primitive hexaploid wheat resource distributed only in Tibet, Tibetan semi-wild wheat (Triticum aestivum subsp. tibetanum Shao) possesses unique characteristics that could be exploited in wheat breeding programs. Its good root system could offer a stable platform for above-ground components. To detect possible excellent locus for root traits from Tibetan semi-wild wheat, we identified QTLs for root traits using a recombinant inbred line population derived from a cross between Tibetan semi-wild wheat Q1028 and Zhengmai 9023. A total of 15 QTLs on eight chromosomes were detected, including four major QTLs, QMrl.sau-7B, QTrl.sau-4B, QAd.sau-7A, and QSa.sau-4B. The phenotypic variation explained by each of these QTLs ranges from 5.67% to 16.68%. Positive alleles of six QTLs were derived from Q1028. Several novel QTLs for root traits were identified. In addition, significant correlations were detected amongst root traits and agronomic traits. Taken together, these results suggest that Tibetan semi-wild wheat and the newly identified novel QTLs could be useful in future breeding programs.

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