The development of CRISPR–Cas systems has sparked a genome editing revolution in plant genetics and breeding. These sequence-specific RNA-guided nucleases can induce DNA double-stranded breaks, resulting in mutations by imprecise non-homologous end joining (NHEJ) repair or precise DNA sequence replacement by homology-directed repair (HDR). However, HDR is highly inefficient in many plant species, which has greatly limited precise genome editing in plants. To fill the vital gap in precision editing, base editing and prime editing technologies have recently been developed and demonstrated in numerous plant species. These technologies, which are mainly based on Cas9 nickases, can introduce precise changes into the target genome at a single-base resolution. This Review provides a timely overview of the current status of base editors and prime editors in plants, covering both technological developments and biological applications. This Review summarizes the current status of base editors and prime editors in plants, reporting both technological developments and biological applications of these precise genome editors.

Precise plant genome editing using base editors and prime editors.

CRISPR/Cas: A powerful tool for gene function study and crop improvement.

Genome- and transcriptome-wide association studies provide insights into the genetic basis of natural variation of seed oil content in Brassica napus.

Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations.

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CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement.

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.

Yellow seed is a desirable trait with great potential for improving seed quality in Brassica crops. Unfortunately, no natural or induced yellow seed germplasms have been found in Brassica napus, an important oil crop, which likely reflects its genome complexity and the difficulty of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we demonstrate the first application of CRISPR/Cas9 for creating yellow-seeded mutants in rapeseed. The targeted mutations of the BnTT8 gene were stably transmitted to successive generations, and a range of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping. The yellow-seeded phenotype could be recovered only in targeted mutants of both BnTT8 functional copies, indicating that the redundant roles of BnA09.TT8 and BnC09.TT8b are vital for seed colour. The BnTT8 double mutants produced seeds with elevated seed oil and protein content and altered fatty acid (FA) composition without any serious defects in the yield-related traits, making it a valuable resource for rapeseed breeding programmes. Chemical staining and histological analysis showed that the targeted mutations of BnTT8 completely blocked the proanthocyanidin (PA)-specific deposition in the seed coat. Further, transcriptomic profiling revealed that the targeted mutations of BnTT8 resulted in the broad suppression of phenylpropanoid/flavonoid biosynthesis genes, which indicated a much more complex molecular mechanism underlying seed colour formation in rapeseed than in Arabidopsis and other Brassica species. In addition, gene expression analysis revealed the possible mechanism through which BnTT8 altered the oil content and fatty acid composition in seeds.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.13281

Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L.

Manipulation of flowering time to develop cultivars with desired maturity dates is fundamental in plant breeding. It is desirable to generate polyploid rapeseed (Brassica napus L.) germplasm with varying flowering time controlled by a few genes. In the present study, BnaSVP, a rapeseed homolog of the Arabidopsis SVP (Short Vegetative Phase) gene, was characterized and a set of mutants was developed using a CRISPR/Cas9-based gene-editing tool. A single construct targeting multiple sites was successfully applied to precisely mutate four copies of BnaSVP. The induced mutations in these copies were stably transmitted to subsequent generations. Homozygous mutants with loss-of-function alleles and free transgenic elements were generated across the four BnaSVP homologs. All mutant T1 lines tested in two environments (summer and winter growing seasons) showed early-flowering phenotypes. The decrease in flowering time was correlated with the number of mutated BnaSVP alleles. The quadruple mutants showed the shortest flowering time, with a mean decrease of 40.6%–50.7% in length relative to the wild type under the two growth conditions. Our study demonstrates the quantitative involvement of BnaSVP copies in the regulation of flowering time and provides valuable resources for rapeseed breeding.

Development of mutants with varying flowering times by targeted editing of multiple SVP gene copies in Brassica napus L.

A major QTL controlling ovule abortion and SN was fine-mapped to a
80.1-kb region on A8 in rapeseed, and BnaA08g07940D and BnaA08g07950D are the most likely candidate genes.
 The seed number per silique (SN), an important yield determining trait of rapeseed, is the final consequence of a complex developmental process including ovule initiation and the subsequent ovule/seed development. To explore the genetic mechanism regulating the natural variation of SN and its related components, quantitative trait locus (QTL) mapping was conducted using a doubled haploid (DH) population derived from the cross between C4-146 and C4-58B, which showed significant differences in SN and aborted ovule number (AON), but no obvious differences in ovule number (ON). QTL analysis identified 19 consensus QTLs for six SN-related traits across three environments. A novel QTL on chromosome A8, un.A8, which associates with multiple traits, except for ON, was stably detected across the three environments. This QTL explained more than 50% of the SN, AON and percentage of aborted ovules (PAO) variations as well as a moderate contribution on silique length (SL) and thousand seed weight (TSW). The C4-146 allele at the locus increases SN and SL but decreases AON, PAO and TSW. Further fine mapping narrowed down this locus into an 80.1-kb interval flanked by markers BM1668 and BM1672, and six predicted genes were annotated in the delimited region. Expression analyses and DNA sequencing showed that two homologs of Arabidopsis photosystem I subunit F (BnaA08g07940D) and zinc transporter 10 precursor (BnaA08g07950D) were the most promising candidate genes underlying this locus. These results provide a solid basis for cloning un.A8 to reduce the ovule abortion and increase SN in the yield improvement of rapeseed.

/pdf/fine-mapping-and-candidate-gene-analysis-of-a-major-locus-1t648pjz1c.pdf

Fine mapping and candidate gene analysis of a major locus controlling ovule abortion and seed number per silique in Brassica napus L.

The diversity of petal and leaf color can improve the ornamental value of rapeseed and promote the development of agriculture and tourism. The two copies of carotenoid isomerase gene (BnaCRTISO) in Brassica napus (BnaA09.CRTISO and BnaC08.CRTISO) was edited using the CRISPR/Cas9 system in the present study. The mutation phenotype of creamy white petals and yellowish leaves could be recovered only in targeted mutants of both BnaCRTISO functional copies, indicating that the redundant roles of BnaA09.CRTISO and BnaC08.CRTISO are vital for the regulation of petal and leaf color. The carotenoid content in the petals and leaves of the BnaCRTISO double mutant was significantly reduced. The chalcone content, a vital substance that makes up the yellow color, also decreased significantly in petals. Whereas, the contents of some carotenes (lycopene, α-carotene, γ-carotene) were increased significantly in petals. Further, transcriptome analysis showed that the targeted mutation of BnaCRTISO resulted in the significant down-regulation of important genes BnaPSY and BnaC4H in the carotenoid and flavonoid synthesis pathways, respectively; however, the expression of other genes related to carotenes and xanthophylls synthesis, such as BnaPDS3, BnaZEP, BnaBCH1 and BCH2, was up-regulated. This indicates that the molecular mechanism regulating petal color variation in B. napus is more complicated than those reported in Arabidopsis and other Brassica species. These results provide insight into the molecular mechanisms underlying flower color variation in rapeseed and provides valuable resources for rapeseed breeding.

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Site-Directed Mutagenesis of the Carotenoid Isomerase Gene BnaCRTISO Alters the Color of Petals and Leaves in 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.

/pdf/precision-genome-engineering-through-cytidine-base-editing-2h70p7umvm.pdf

Kaidi Yu

Papers

Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L.

Development of mutants with varying flowering times by targeted editing of multiple SVP gene copies in Brassica napus L.

Fine mapping and candidate gene analysis of a major locus controlling ovule abortion and seed number per silique in Brassica napus L.

Site-Directed Mutagenesis of the Carotenoid Isomerase Gene BnaCRTISO Alters the Color of Petals and Leaves in Brassica napus L.

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