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.

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 A site-directed mutagenesis study of human placental aromatase.

1992 ◽  
Vol 267 (2) ◽  
pp. 762-768
Author(s):  
D J Zhou ◽  
K R Korzekwa ◽  
T Poulos ◽  
S A Chen
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
A Site ◽  
Mutagenesis Study

scholarly journals Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

1997 ◽  
Vol 109 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Laurent Schild ◽  
Estelle Schneeberger ◽  
Ivan Gautschi ◽  
Dmitri Firsov
Keyword(s):  
Amino Acid ◽  
Ion Selectivity ◽  
Site Directed Mutagenesis ◽  
Ion Permeation ◽  
Amino Acid Residues ◽  
Α Subunit ◽  
Na Ions ◽  
Membrane Spanning ◽  
A Site ◽  
Epithelial Sodium Channel Enac

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (Ki) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn2+ ions, in particular the αS583C mutant showing a Ki for Zn2+of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride Ki, the later mutation generating a Ca2+blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ions at an external Na+binding site preventing ion permeation through the channel pore.

Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae

1993 ◽  
Vol 13 (12) ◽  
pp. 7836-7849
Author(s):  
P Russo ◽  
W Z Li ◽  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
In Vitro Mutagenesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Element ◽  
A Site ◽  
Derivatives Of ◽  
Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

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.

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