Genome‐wide analyses of PAM‐relaxed Cas9 genome editors reveal substantial off‐target effects by ABE8e in rice

Wu, Yuechao; Ren, Qiao; Zhong, Zhiyong; Liu, Guanqing; Han, Yangshuo; Bao, Yu; Liu, Li; Xiang, Shuyue; Liu, Shuo; Tang, Xu; Zhou, Jianping; Zheng, Xuelian; Sretenovic, Simon; Zhang, Tao; Qi, Yiping; Zhang, Yong

doi:10.1111/pbi.13838

Cited by 33 publications

(33 citation statements)

References 73 publications

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“…The results showed that the efficiency and stability of the CRISPR‐Cas12a system were higher than that of the CRISPR‐Cas9 system. This was consistent with previous reports (Tang et al., 2017; Wu et al., 2022). We performed genotype analysis on partial mutants, and the results showed that although Cas9 system also had large fragment deletions, most of the mutations of Cas9 system were 1–3 bp InDel (insertions and deletions), especially 1 bp InDel.…”

Section: Resultssupporting

confidence: 94%

Loss‐function mutants of OsCKX gene family based on CRISPR‐Cas systems revealed their diversified roles in rice

et al. 2023

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Cytokinin (CTK) is an important plant hormone that promotes cell division, controls cell differentiation, and regulates a variety of plant growth and development processes. Cytokinin oxidase/dehydrogenase (CKX) is an irreversible cytokinindegrading enzyme that affects plant growth and development by regulating the dynamic balance of CTKs synthesis and degradation. There are presumed 11 members of the CKX gene family in rice (Oryza sativa L.), but limited members have been reported. In this study, based on CRISPR-Cas9 and CRISPR-Cas12a genomeediting technology, we established a complete set of OsCKX1-OsCKX11 single-gene mutants, as well as double-gene and triple-gene mutants of different OsCKXs gene combinations with high similarity. The results revealed that CRISPR-Cas12a outperformed Cas9 to generate biallelic mutations, multi-gene mutants, and more diverse genotypes. And then, we found, except the reported OsCKX2, OsCKX4, OsCKX9 and OsCKX11, OsCKX5, OsCKX6, OsCKX7, and OsCKX8 also had significant effects on agronomic traits such as plant height, panicle size, grain size, and grain number per panicle in rice. In addition, the different loss-of-function of the OsCKX genes also changed the seed appearance quality and starch composition. Interestingly, by comparing different combinations of multi-gene mutants, we found significant functional redundancy among OsCKX gene members in the same phylogenetic clade. These data collectively reveal the diversified regulating capabilities of OsCKX genes in rice, and also provide the valuable reference for further rice molecular breeding. INTRODUCTIONCytokinin (CTK) is a class of plant hormone that contains adenine derivatives and plays a key role in plant growth and development. Cytokinin has been found to be involved in promoting cell division, regulating cell differentiation,

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Section: Resultssupporting

confidence: 94%

Loss‐function mutants of OsCKX gene family based on CRISPR‐Cas systems revealed their diversified roles in rice

et al. 2023

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“…S2 ). Surprisingly, PmCDA1-derived NG-CBE showed no self-editing for both the tested sites similar to a recent report in plants ( 32 ). These results indicated that regardless of self-editing, nCas9-NG-based BEs enable base editing in E. coli .…”

Section: Resultssupporting

confidence: 90%

“…In the case of the PmCDA1-based CBE-NG tool, we did not observe self-editing in bacteria which is consistent with the previous reports of PmCDA1-based CBEs with nCas9 ( 40 ), Cas9-NG, and SpRY ( 32 ) in plant systems. Hence, when desirable, we propose PmCDA1 as a first-choice CBE with PAM-relaxed Cas9 variants.…”

Section: Discussionsupporting

confidence: 92%

Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli

Shelake

Pramanik

Kim

2023

mBio

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“…The results suggest that the mutation type may not be a decisive factor for determining the augmented seed oleic acid content. Although genome editing with the CRISPR/Cas9 system is an efficient method in crop trait improvement, the system has a risk of off-target resulting undesirable mutations because of the similar sequences between on-target and off-target sites ( Park et al., 2022 ; Wu et al., 2022 ). Many strategies were attempted to reduce genome-wide off-target events and made great achievements ( Fu et al., 2014 ; Kleinstiver et al., 2016 ; Zhang et al., 2019 ).…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated editing of double loci of BnFAD2 increased the seed oleic acid content of rapeseed (Brassica napus L.)

et al. 2022

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Seed oleic acid is an important quality trait sought in rapeseed breeding programs. Many methods exist to increase seed oleic acid content, such as the CRISPR/Cas9-mediated genome editing system, yet there is no report on seed oleic acid content improvement via this system’s precise editing of the double loci of BnFAD2. Here, a precise CRISPR/Cas9-mediated genome editing of the encoded double loci (A5 and C5) of BnFAD2 was established. The results demonstrated high efficiency of regeneration and transformation, with the rapeseed genotype screened in ratios of 20.18% and 85.46%, respectively. The total editing efficiency was 64.35%, whereas the single locus- and double locus-edited ratios were 21.58% and 78.42%, respectively. The relative proportion of oleic acid with other fatty acids in seed oil of mutants was significantly higher for those that underwent the editing on A5 copy than that on C5 copy, but it was still less than 80%. For double locus-edited mutants, their relative proportion of oleic acid was more than 85% in the T1 and T4 generations. A comparison of the sequences between the double locus-edited mutants and reference showed that no transgenic border sequences were detected from the transformed vector. Analysis of the BnFAD2 sequence on A5 and C5 at the mutated locus of double loci mutants uncovered evidence for base deletion and insertion, and combination. Further, no editing issue of FAD2 on the copy of A1 was detected on the three targeted editing regions. Seed yield, yield component, oil content, and relative proportion of oleic acid between one selected double loci-edited mutant and wild type were also compared. These results showed that although the number of siliques per plant of the wild type was significantly higher than those of the mutant, the differences in seed yield and oil content were not significant between them, albeit with the mutant having a markedly higher relative proportion of oleic acid. Altogether, our results confirmed that the established CRISPR/Cas9-mediated genome editing of double loci (A5 and C5) of the BnFAD2 can precisely edit the targeted genes, thereby enhancing the seed oleic acid content to a far greater extent than can a single locus-editing system.

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Genome‐wide analyses of PAM‐relaxed Cas9 genome editors reveal substantial off‐target effects by ABE8e in rice

Cited by 33 publications

References 73 publications

Loss‐function mutants of OsCKX gene family based on CRISPR‐Cas systems revealed their diversified roles in rice

Loss‐function mutants of OsCKX gene family based on CRISPR‐Cas systems revealed their diversified roles in rice

Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli

CRISPR/Cas9-mediated editing of double loci of BnFAD2 increased the seed oleic acid content of rapeseed (Brassica napus L.)

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