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.

Genome engineering for crop improvement and future agriculture

Empirical threshold values for quantitative trait mapping.

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

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

Sequence-specific nucleases (SSN) that generate double-stranded DNA breaks (DSBs) in genes of interest are the key to site-specific genome editing in plants. Genome editing has developed into one method of reducing undesirable traits in crops by the induction of knockout mutations. Different SSN-mediated genome-editing systems, including LAGLIDADG homing endonucleases or meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats, are emerging as robust tools for introducing functional mutations in polyploid crops including citrus, wheat, cotton, soybean, rapeseed, potato, grapes, Camelina sativa, dandelion, and tobacco. The approach utilizes knowledge of biological mechanisms for targeted induction of DSBs and their error-prone repair, allowing highly specific changes at designated genome loci. In this review, we briefly describe genome-editing technologies and their application to genetic improvement of polyploid crops.

Genome editing opens a new era of genetic improvement in polyploid crops

CRISPR/Cas-base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A-PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base-editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base-edited plants of ALS conferred high herbicide resistance, while base-edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T0 to T1 generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off-target sites, large-scale SNP variations were determined through whole-genome sequencing between some base-edited and wild-type plants. These results revealed that A3A-PBE base-editing system could effectively convert C to T substitution with high-editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13444

Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system.

Oilseed rape (Brassica napus L.) is one of the most important oil crops in China as well as worldwide. Branch angle as a plant architecture component trait plays an important role for high density planting and yield performance. In this study, bulked segregant analysis (BSA) combined with next generation sequencing technology was used to fine map QTL for branch angle. A major QTL, designated as branch angle 1 (ba1) was identified on A06 and further validated by Indel marker-based classical QTL mapping in an F2 population. Eighty-two genes were identified in the ba1 region. Among these genes, BnaA0639380D is a homolog of AtYUCCA6. Sequence comparison of BnaA0639380D from small- and big-branch angle oilseed rape lines identified six SNPs and four amino acid variation in the promoter and coding region, respectively. The expression level of BnaA0639380D is significantly higher in the small branch angle line Purler than in the big branch angle line Huyou19, suggesting that the genomic mutations may result in reduced activity of BnaA0639380D in Huyou19. Phytohormone determination showed that the IAA content in Purler was also obviously increased. Taken together, our results suggested BnaA0639380D is a possible candidate gene for branch angle in oilseed rape.

/pdf/identification-of-bnayucca6-as-a-candidate-gene-for-branch-4455m1baoo.pdf

Identification of BnaYUCCA6 as a candidate gene for branch angle in Brassica napus by QTL-seq.

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.

Hongtao Cheng

Papers

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

Genome editing opens a new era of genetic improvement in polyploid crops

Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system.

Identification of BnaYUCCA6 as a candidate gene for branch angle in Brassica napus by QTL-seq.

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