A sequential transformation method for validating soybean genome editing by CRISPR/Cas9 system

Trinh, Duy Dinh; Le, Ngoc Thu; Bui, Thao Phuong; Le, Thao Nhu Thi; Nguyen, Cuong Xuan; Chu, Ha Hoang; Tien, Phat

doi:10.1016/j.sjbs.2022.103420

Cited by 5 publications

(2 citation statements)

References 36 publications

(61 reference statements)

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“…In our study, many types of mutation were found. Multiple mutation forms were also found in other investigations ( Okuzaki et al., 2018 ; Huang et al., 2020 ; Trinh et al., 2022 ). The results suggest that the mutation type may not be a decisive factor for determining the augmented seed oleic acid content.…”

Section: Discussionsupporting

confidence: 82%

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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Section: Discussionsupporting

confidence: 82%

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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“…In 2020, He et al coupled a CRISPR/Cas9 cassette with a unit that activated anthocyanin biosynthesis to provide a screening marker for visibility. In 2022, Trinh et al screened transgenic hairy roots by adding a GFP fluorescent marker to the editing vector [ 178 ].…”

Section: Strategies and Methods For Optimizing Crispr/cas9 Gene-editi...mentioning

confidence: 99%

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Zhou

Luan

Liu

et al. 2023

Plants

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Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature and is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation of transgene-free genome-edited plants in a short period and has the advantages of simplicity, high efficiency, high specificity, and low production costs, which greatly facilitate the study of gene functions. In plant molecular breeding, the gene-editing efficiency of the CRISPR-Cas9 system has proven to be a key step in influencing the effectiveness of molecular breeding, with improvements in gene-editing efficiency recently becoming a focus of reported scientific research. This review details strategies and methods for improving the efficiency of CRISPR/Cas9 gene editing in plant molecular breeding, including Cas9 variant enzyme engineering, the effect of multiple promoter driven Cas9, and gRNA efficient optimization and expression strategies. It also briefly introduces the optimization strategies of the CRISPR/Cas12a system and the application of BE and PE precision editing. These strategies are beneficial for the further development and optimization of gene editing systems in the field of plant molecular breeding.

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated Proteins (Cas) [CRISPR–Cas]: An Emerging Technique in Plant Disease Detection and Management

Ghosh,

Chatterjee

2024

Gene Editing in Plants

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A sequential transformation method for validating soybean genome editing by CRISPR/Cas9 system

Cited by 5 publications

References 36 publications

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

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

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated Proteins (Cas) [CRISPR–Cas]: An Emerging Technique in Plant Disease Detection and Management

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