The availability of genome sequences for several crops and advances in genome editing approaches has opened up possibilities to breed for almost any given desirable trait. Advancements in genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) has made it possible for molecular biologists to more precisely target any gene of interest. However, these methodologies are expensive and time-consuming as they involve complicated steps that require protein engineering. Unlike first-generation genome editing tools, CRISPR/Cas9 genome editing involves simple designing and cloning methods, with the same Cas9 being potentially available for use with different guide RNAs targeting multiple sites in the genome. After proof-of-concept demonstrations in crop plants involving the primary CRISPR-Cas9 module, several modified Cas9 cassettes have been utilized in crop plants for improving target specificity and reducing off-target cleavage (eg. Nmcas9, Sacas9, Stcas9). Further, the availability of Cas9 enzymes from additional bacterial species has made available options to enhance specificity and efficiency of gene editing methodologies. This review summarizes the options available to plant biotechnologists to bring about crop improvement using CRISPR/Cas9 based genome editing tools and also presents studies where CRISPR/Cas9 has been used for enhancing biotic and abiotic stress tolerance. Application of these techniques will result in the development of non-genetically modified (Non-GMO) crops with the desired trait that can contribute to increased yield potential under biotic and abiotic stress conditions.

/pdf/crispr-for-crop-improvement-an-update-review-53da0nv2w2.pdf

CRISPR for Crop Improvement: An Update Review.

https://escholarship.org/content/qt5ps288gh/qt5ps288gh.pdf?t=p68yl9

Central role of dysregulation of TGF-β/Smad in CKD progression and potential targets of its treatment

CRISPR/Cas System: Recent Advances and Future Prospects for Genome Editing

In most crop breeding programs, the rate of yield increment is insufficient to cope with the increased food demand caused by a rapidly expanding global population. In plant breeding, the development of improved crop varieties is limited by the very long crop duration. Given the many phases of crossing, selection, and testing involved in the production of new plant varieties, it can take one or two decades to create a new cultivar. One possible way of alleviating food scarcity problems and increasing food security is to develop improved plant varieties rapidly. Traditional farming methods practiced since quite some time have decreased the genetic variability of crops. To improve agronomic traits associated with yield, quality, and resistance to biotic and abiotic stresses in crop plants, several conventional and molecular approaches have been used, including genetic selection, mutagenic breeding, somaclonal variations, whole-genome sequence-based approaches, physical maps, and functional genomic tools. However, recent advances in genome editing technology using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) proteins have opened the door to a new plant breeding era. Therefore, to increase the efficiency of crop breeding, plant breeders and researchers around the world are using novel strategies such as speed breeding, genome editing tools, and high-throughput phenotyping. In this review, we summarize recent findings on several aspects of crop breeding to describe the evolution of plant breeding practices, from traditional to modern speed breeding combined with genome editing tools, which aim to produce crop generations with desired traits annually.

https://www.mdpi.com/1422-0067/21/7/2590/pdf

Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T0 plants were edited very efficiently (56.30%-67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi-allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T1 and T2 progeny. We also obtained transgene-free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off-target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi-dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi-dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.13228

Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.).

With the rapid development of sequence specific nucleases (SSNs) for genome targeting, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is now considered the most promising method for functional genetic researches, as well as genetic improvement in crop plants. However, the gene redundancy feature within the allotetraploid rapeseed genome is one of the major obstacles for simultaneous modification of different homologs in the first generation. In addition, large scale screening to identify mutated transgenic plants is very time-and labor-consuming using the conventional restriction enzyme-based approaches. In this study, a streamlined rapeseed CRISPR-Cas9 genome editing platform was developed through synthesizing a premade U6-26 driven sgRNA expression cassette and optimizing polyacrylamide gel electrophoresis (PAGE)-based screening approach. In our experiment, a sgRNA was constructed to target five rapeseed SPL3 homologous gene copies, BnSPL3-A5/BnSPL3-A4/BnSPL3-C3/BnSPL3-C4/BnSPL3-Cnn. High-throughput sequencing analysis demonstrated that the editing frequency of CRISPR/Cas9-induced mutagenesis ranged from 96.8 to 100.0% in plants with obvious heteroduplexed PAGE bands, otherwise this proportion was only 0.00-60.8%. Consistent with those molecular analyses, Bnspl3 mutants exhibited developmental delay phenotype in the first generation. In summary, our data suggest that this set of CRISPR/Cas9 platform is qualified for rapidly generating and identifying simultaneous mutagenesis of multiple gene homologs in allotetraploid rapeseed.

/pdf/an-efficient-crispr-cas9-platform-for-rapidly-generating-1c3dhyu1pt.pdf

An Efficient CRISPR/Cas9 Platform for Rapidly Generating Simultaneous Mutagenesis of Multiple Gene Homoeologs in Allotetraploid Oilseed Rape

Pod shattering resistance is an essential component to achieving a high yield, which is a substantial objective in polyploid rapeseed cultivation. Previous studies have suggested that the Arabidopsis JAGGED (JAG) gene is a key factor implicated in the regulatory web of dehiscence fruit. However, its role in controlling pod shattering resistance in oilseed rape is still unknown. In this study, multiplex genome editing was carried out by the CRISPR/Cas9 system on five homoeologs (BnJAG.A02, BnJAG.C02, BnJAG.C06, BnJAG.A07, and BnJAG.A08) of the JAG gene. Knockout mutagenesis of all homoeologs drastically affected the development of the lateral organs in organizing pod shape and size. The cylindrical body of the pod comprised a number of undifferentiated cells like a callus, without distinctive valves, replum, septum, and valve margins. Pseudoseeds were produced, which were divided into two halves with an incomplete layer of cells (probably septum) that separated the undifferentiated cells. These mutants were not capable of generating any productive seeds for further generations. However, one mutant line was identified in which only a BnJAG.A08-NUB-Like paralog of the JAG gene was mutated. Knockout mutagenesis in BnJAG.A08-NUB gene caused significant changes in the pod dehiscence zone. The replum region of the mutant was increased to a great extent, resulting in enlarged cell size, bumpy fruit, and reduced length compared with the wild type. A higher replum–valve joint area may have increased the resistance to pod shattering by ~2-fold in JAG mutants compared with wild type. Our results offer a basis for understanding variations in Brassica napus fruit by mutating JAG genes and providing a way forward for other Brassicaceae species.

CRISPR/Cas9-Mediated Multiplex Genome Editing of JAGGED Gene in Brassica napus L.

Computational tools and resources for CRISPR/Cas genome editing.

Oilseed rape (Brassica napus L.) is the second largest oilseed crop worldwide and one of the most important oil crops in China. As a component of plant architecture, branch angle plays an important role in yield performance, especially under high-density planting conditions. However, the mechanisms underlying the regulation of branch angle are still largely not understood. Two oilseed rape lines with significantly different branch angles were used to conduct RNA- and miRNA-profiling at two developmental stages, identifying differential expression of a large number of genes involved in auxin- and brassinosteroid (BR)-related pathways. Many auxin response genes, including AUX1, IAA, GH3, and ARF, were enriched in the compact line. However, a number of genes involved in BR signaling transduction and biosynthesis were down-regulated. Differentially expressed miRNAs included those involved in auxin signaling transduction. Expression patterns of most target genes were fine-tuned by related miRNAs, such as miR156, miR172, and miR319. Some miRNAs were found to be differentially expressed at both developmental stages, including three miR827 members. Our results provide insight that auxin- and BR-signaling may play a pivotal role in branch angle regulation.

https://www.mdpi.com/1422-0067/18/5/887/pdf

Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13632

Wen Chu

Papers

An Efficient CRISPR/Cas9 Platform for Rapidly Generating Simultaneous Mutagenesis of Multiple Gene Homoeologs in Allotetraploid Oilseed Rape

CRISPR/Cas9-Mediated Multiplex Genome Editing of JAGGED Gene in Brassica napus L.

Computational tools and resources for CRISPR/Cas genome editing.

Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus.

Direct modification of multiple gene homoeologs in Brassica oleracea and Brassica napus using doubled haploid inducer-mediated genome-editing system.