scholarly journals Establishment of A PEG-mediated Protoplast Transformation System Based on DNA and CRISPR/Cas9 Ribonucleoprotein Complexes For Banana

2020 ◽  
Author(s):  
Shaoping Wu ◽  
Haocheng Zhu ◽  
Jinxing Liu ◽  
Qiaosong Yang ◽  
Xiuhong Shao ◽  
...  
Keyword(s):  
Transient Expression ◽  
Amplicon Sequencing ◽  
Enzyme Digestion ◽  
Site Directed Mutagenesis ◽  
Plant Science ◽  
Restriction Enzyme Digestion ◽  
Protoplast Transformation ◽  
Transformation System ◽  
Ribonucleoprotein Complexes ◽  
Target Sites

Abstract Background: To date, CRISPR/Cas9 RNPs editing tools have not been applied to genetic modification of banana. Here, the establishment of PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA- free method for the site-directed mutagenesis system.Results: Protoplasts are a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering plasmids of CRISPR/Cas9 and CRISPR/Cas12a , CRISPR/Cas9 ribonucleoproteins (RNPs) that targeted PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays and monoclonal sequencing further confirmed the occurring of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of CRISPR/Cas9 system was higher than that of the other two systems. Conclusions: The PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana.The application of CRISPR/Cas9 RNPs system enables the generation of DNA-free genome edited banana plants.

scholarly journals Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Shaoping Wu ◽  
Haocheng Zhu ◽  
Jinxing Liu ◽  
Qiaosong Yang ◽  
Xiuhong Shao ◽  
...  
Keyword(s):  
Transient Expression ◽  
Targeted Delivery ◽  
Amplicon Sequencing ◽  
Site Directed Mutagenesis ◽  
Restriction Enzyme Digestion ◽  
Protoplast Transformation ◽  
Transformation System ◽  
Ribonucleoprotein Complexes ◽  
Free Gene ◽  
A Site

Abstract Background To date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system. Results Protoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of the PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems. Conclusions The PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.

The stability of maize nuclei to restriction enzyme digestion differs with transcriptional activity

Caryologia ◽  
1993 ◽  
Vol 46 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Valeria Mirkova ◽  
Maria Ivanchenko ◽  
Lubomir Stoilov ◽  
Jordanka Zlatanova
Keyword(s):  
Restriction Enzyme ◽  
Transcriptional Activity ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
The Stability

scholarly journals Biolistic-mediated transient gene expression in shoot apical meristems of the prickly-pear (Opuntia ficus-indica)

1999 ◽  
Vol 42 (3) ◽  
pp. 299-302 ◽  
Author(s):  
Romulo Marino Llamoca-Zárate ◽  
Luiz Ferreira Aguiar Ponte ◽  
Joerg Landsmann ◽  
Francisco de Assis Paiva Campos
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Transient Gene Expression ◽  
Dna Delivery ◽  
Prickly Pear ◽  
Target Tissue ◽  
Gus Gene ◽  
Transformation System ◽  
Opuntia Ficus Indica ◽  
Apical Meristems

We have demonstrated the transient expression of the GUS gene in cells of the meristematic apical dome of Opuntia ficus-indica. DNA delivery into the cells was achieved using a biolistic PDS-1000He instrument from Bio-Rad Laboratories. The transforming DNA was coated in tungsten particles with diameter of 1.3 m m and the distance between the flying disk and the target tissue was 7.5cm and the shooting pressure was adjusted to 1200 psi. This is the first demonstration that the biolistic transformation system can be used to express a transgene in a member of the Cactaceae.

scholarly journals A novel decatenation assay for DNA topoisomerases using a singly-linked catenated substrate

BioTechniques ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 356-362
Author(s):  
Nidda F Waraich ◽  
Shruti Jain ◽  
Sean D Colloms ◽  
William Marshall Stark ◽  
Nicolas P Burton ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Kinetoplast Dna ◽  
Dna Topoisomerases ◽  
In Vitro Drug Screening ◽  
Drug Inhibition ◽  
Dna Substrate

Decatenation is a crucial in vivo reaction of DNA topoisomerases in DNA replication and is frequently used in in vitro drug screening. Usually this reaction is monitored using kinetoplast DNA as a substrate, although this assay has several limitations. Here we have engineered a substrate for Tn 3 resolvase that generates a singly-linked catenane that can readily be purified from the DNA substrate after restriction enzyme digestion and centrifugation. We show that this catenated substrate can be used with high sensitivity in topoisomerase assays and drug-inhibition assays.

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