Engineering herbicide‐resistant oilseed rape by CRISPR/Cas9‐mediated cytosine base‐editing

Wu, Jian; Chen, Chen; Xian, Guiyu; Liu, Dongxiao; Li, Lin; Yin, Shengliang; Sun, Qinfu; Fang, Yujie; Zhang, Hui; Wang, Youping

doi:10.1111/pbi.13368

Cited by 92 publications

(63 citation statements)

References 9 publications

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“…The plants harboured substitutions at C1, C6, C7, C8 and C10 sites, indicated that the deamination editing window could range from 1 to 10 base sites. As BE3 system was demonstrated to efficiently edit single base within approximately a five-nucleotide window of À16 to À12 bp from the PAM sequence (with deamination editing window from C4 to C8) (Wu et al, 2020), our results showed A3A-PBE system is more efficient to conduct base editing in allotetraploid B. napus. Among all the 11 edited plants of BnaALS, each one harboured at least three site base substitutions simultaneously.…”

Section: Target Mutation Analysis Of Transgenic Plants For Als Genementioning

confidence: 70%

“…Genes controlling important agronomic traits, such as pod shattering, plant height, seed coat colour and silique locular, have been modified using CRISPR/Cas9 method (Li et al, 2018c;Yang et al, 2017;Yang et al, 2018;Zaman et al, 2019;Zhai et al, 2019 andZheng et al, 2020 ). One baseediting system, which is consisted of nCas9, rat cytidine deaminase and a uracil glycosylase inhibitor, has been established in Brassicaceae (Wu et al, 2020). Four plants of BnaC01.ALS were detected to contain effective base variation at the target site, and the editing efficiency was approximately 1.8% (Wu et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…One baseediting system, which is consisted of nCas9, rat cytidine deaminase and a uracil glycosylase inhibitor, has been established in Brassicaceae (Wu et al, 2020). Four plants of BnaC01.ALS were detected to contain effective base variation at the target site, and the editing efficiency was approximately 1.8% (Wu et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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

Cheng

Hao

Ding

et al. 2020

Plant Biotechnology Journal

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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 T 0 to T 1 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.

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Section: Target Mutation Analysis Of Transgenic Plants For Als Genementioning

confidence: 70%

Section: Introductionmentioning

confidence: 99%

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

Cheng

Hao

Ding

et al. 2020

Plant Biotechnology Journal

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“…Both CBE and ABE have been successfully applied in various model plants and crops ( Chen et al., 2019 ). They are widely used to introduce targeted substitutions in major genes to improve important agricultural traits, including plant height, flowering time, disease resistance, and herbicide resistance ( Chen et al., 2017 , Lu and Zhu, 2017 , Shimatani et al., 2017 , Kang et al., 2018 , Li et al., 2018 , Li et al., 2019a , Tian et al., 2018 , Bastet et al., 2019 , Zhang et al., 2019 , Wu et al., 2020 ). Base editors were also employed to disrupt genes in plants by creating early stop codons or inducing transcript mis-splicing ( Kang et al., 2018 , Xue et al., 2018 , Li et al., 2019b , Veillet et al., 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Development of Plant Prime-Editing Systems for Precise Genome Editing

Liu

et al. 2020

Plant Communications

165

123

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Prime-editing systems have the capability to perform efficient and precise genome editing in human cells. In this study, we first developed a plant prime editor 2 (pPE2) system and test its activity by generating a targeted mutation on an HPT -ATG reporter in rice. Our results showed that the pPE2 system could induce programmable editing at different genome sites. In transgenic T 0 plants, pPE2-generated mutants occurred with 0%–31.3% frequency, suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures. To optimize editing efficiency, guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells. However, at the genomic sites tested in this study, pPE3 systems generated only comparable or even lower editing frequencies. Furthemore, we developed a surrogate pPE2 system by incorporating the HPT -ATG reporter to enrich the prime-edited cells. The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system, presumably due to the enhanced screening efficiency of edited cells. Taken together, our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.

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“…The improved BnA3A1-PBE performed efficiently as a base editor with higher editing efficiency, a more broadened editing window coupled with a higher proportion of homozygous and biallelic genotypes compared with BnA3A-PBE and BnPBE systems. When compared with the two latest studies which reported the successful application of cytosine base-editing in rapeseed (Wu et al, 2020, Cheng et al, 2020, BnA3A1-PBE has a high editing efficiency, which is ∼16-fold than PBE (Wu et al, 2020), and 26% higher than those of A3A-PBE (Cheng et al, 2020). Thus, BnA3A1-PBE represents a powerful base editor for both gene function study and molecular breeding in rapeseed.…”

Section: The Ng Protospacer Adjacent Motif Greatly Broaden the Targetmentioning

confidence: 91%

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

Amoo

Liu

et al. 2020

Front. Genome Ed.

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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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Engineering herbicide‐resistant oilseed rape by CRISPR/Cas9‐mediated cytosine base‐editing

Cited by 92 publications

References 9 publications

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

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

Development of Plant Prime-Editing Systems for Precise Genome Editing

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

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