A Transformation and Genome Editing System for Cassava Cultivar SC8

Wang, Yajie; Lü, Xiaohua; Zhen, Xing-Hou; Ye, Hui; Che, Yannian; Hou, Jingyi; Geng, Meiyu; Liu, Jiao; Hu, Xinwen; Li, Ruimei; Guo, Jian-Chun; Yao, Yuan

doi:10.3390/genes13091650

Cited by 21 publications

(9 citation statements)

References 41 publications

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“…This indicates that the editing efficiency of the gene editing vector currently used is not efficient, which need to be optimized. The promoter of Cas9 gene have a significant impact not only on editing efficiency, but also have an impact on the editing type, many other promoters produce higher editing efficiency and a higher proportion of homozygous or biallelic mutations than the CaMV35S promoter (Feng et al ., 2018; Wang, et al ., 2022). The Cas9 gene sequence in present study is optimized based on the rice codon, driven by 2 × 35S promoter, next we will replace the 2×35S promoter of Cas9 with the endogenous constitutive strong promoter pHbUbiquitin of H. brasiliensis (Xin, et al ., 2022), and optimize Cas9 gene according to the codon bias of H. brasiliensis .…”

Section: Discussionmentioning

confidence: 99%

An optimized somatic embryo transformation system assisted homozygous edited rubber tree generation method mediated by CRISPR/Cas9

Yang,

Lin,

Udayabhanu

et al. 2024

Preprint

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Previously, we have realized the CRISPR/Cas9-RNP and plasmid mediated protoplast transient transformation genome editing in the rubber tree (Hevea brasiliensis), but no gene editing plants were acquired due to the bottleneck of genetic transformation. In present study, antibiotic sensitivity tests against kanamycin, hygromycin and basta were analyzed for embryo screening, the results demonstrated that 10 mg/L hygromycin is the best for transformation. Then Agrobacterium mediated transformation of H. brasiliensis embryos was carried out using a pCAMBIA1300-based CRISPR/Cas9 vector targeting Phytoene desaturase gene (HbPDS). High-throughput sequencing of T0 generation positive embryos which were used as regeneration materials in typical transformation procedure showed that more than 90% T0 edited embryos are chimeric with a 3.2% transformation efficiency. A T0 embryo with 9.8% edited cells was sliced into small pieces for one more cycle embryogenesis to produce T1 generation embryos in order to improve the ratio of homozygous embryos. Subsequently, next-generation sequencing (NGS) demonstrated that 29 out of 33 T1 embryos were edited, nearly 50% of which were found homozygous. At last, besides four chimeric plantlets with partial albino leaves, four plantlets with complete albino phenotype were regenerated from the 29 T1 generation edited embryos, among which one is a homozygous mono-allelic mutant and the other three are homozygous bi-allelic mutants. NGS demonstrated that the threshold for the proportion of edited cells with expected albino phenotype is between 70-85%. Additionally, Tail-PCR indicate that the T-DNA was inserted into different genome positions in the four homozygous edited plantlets, combined with the different genotypes are considered, the four homozygous plantlets can be confirmed as independently derived from single transformed cells. Overall, this is the first edited rubber trees with expected phenotype reported publicly, which shows the potential in genetic improvement of H. brasiliensis by CRISPR/Cas9 gene editing, and subculture of T0 positive transformed somatic embryos into T1 generation is proved to be an effective and necessary procedure to produce homozygous transgenic plantlets. This study presents a significant advancement in transgenic and gene editing for rubber tree.

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Section: Discussionmentioning

confidence: 99%

An optimized somatic embryo transformation system assisted homozygous edited rubber tree generation method mediated by CRISPR/Cas9

Yang,

Lin,

Udayabhanu

et al. 2024

Preprint

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“…GBVS enhanced mutation screening overall, with a 2.58~7.50-fold increase in frequency, and 25~48.15% of T 1 generation Arabidopsis screened by the GBVS system were homozygous or biallelic mutants, a 1.71~7.86-fold higher proportion than those screened using the original system [ 168 ]. In 2022, Wang et al used the pYAO promoter to drive CRISPR/Cas9 to generate 45.83% homozygous single mutations in the MePDS gene, opening a more functional pathway for the genetic improvement of cassava SC8 [ 169 ]. The use of a germline-specific promoter would have multiple benefits, such as reducing the potential toxicity associated with Cas9 expression under other strong constitutive promoters.…”

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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“…Successful FEC induction has laid a foundation for genetic transformation [28,31,32], CRISPR/Cas9 genome editing technology [33,34], protoplast culture and regeneration [35,36], and somatic hybridization [37].…”

Section: Friable Embryogenic Callus (Fec) Inductionmentioning

confidence: 99%

Plant Regeneration from Cassava Protoplasts

Wen¹,

Fu²,

Luo³

et al. 2024

Cassava - Recent Updates on Food, Feed, and Industry

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Cassava is an important crop for food, feed, and industrial raw materials. Given that traditional conventional breeding is restricted by various factors, biotechnology breeding has become an important breeding method. Tissue culture regeneration is the basis of biotechnology breeding. This chapter reviews the establishment and development of cassava tissue culture and regeneration systems and the technical processes of tissue culture and regeneration starting from the induction of explants of tissue-cultured cassava plantlets to embryogenic calli, isolation to protoplasts, culture to embryogenic calli followed by differentiation into embryos, and then sprouting, stemming, and rooting into complete plants. This chapter focuses on the technical processes from protoplast to complete plant and summarizes the important influencing factors of protoplast regeneration, which is the key and difficult point in the entire regeneration process of cassava protoplasts. This chapter aims to provide technical guidance for cassava protoplast regeneration, offer useful inspiration and reference for cassava tissue culture, and lay a foundation for the genetic improvement of cassava.

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A Transformation and Genome Editing System for Cassava Cultivar SC8

Cited by 21 publications

References 41 publications

An optimized somatic embryo transformation system assisted homozygous edited rubber tree generation method mediated by CRISPR/Cas9

An optimized somatic embryo transformation system assisted homozygous edited rubber tree generation method mediated by CRISPR/Cas9

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

Plant Regeneration from Cassava Protoplasts

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