Faculty Opinions recommendation of Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase.

2013 ◽  
Author(s):  
Thomas Roitsch ◽  
Eric Van Der Graaff
Keyword(s):  
Arabidopsis Thaliana ◽  
Cre Recombinase ◽  
Site Specific ◽  
Dna Integration ◽  
Combined Action

scholarly journals Site-specific T-DNA integration inArabidopsis thalianamediated by the combined action of CRE recombinase and ϕC31 integrase

2013 ◽  
Vol 75 (1) ◽  
pp. 172-184 ◽  
Author(s):  
Annelies De Paepe ◽  
Sylvie De Buck ◽  
Jonah Nolf ◽  
Els Van Lerberge ◽  
Ann Depicker
Keyword(s):  
Cre Recombinase ◽  
Site Specific ◽  
Dna Integration ◽  
Combined Action

scholarly journals Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain

2020 ◽  
Vol 48 (18) ◽  
pp. 10590-10601
Author(s):  
Shufeng Ma ◽  
Xinlong Wang ◽  
Yongfei Hu ◽  
Jie Lv ◽  
Chengfang Liu ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Sleeping Beauty ◽  
Binding Domain ◽  
Protein Fusion ◽  
Site Specific ◽  
Dna Integration ◽  
Cas9 Nuclease ◽  
Human T Cells ◽  
Dna Insertion ◽  
Aavs1 Locus

Abstract The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.

scholarly journals Agrobacterium tumefaciens Transformation of the Radiation Hypersensitive Arabidopsis thaliana Mutants uvh1 and rad5

1998 ◽  
Vol 11 (11) ◽  
pp. 1136-1141 ◽  
Author(s):  
Jaesung Nam ◽  
Kirankumar S. Mysore ◽  
Stanton B. Gelvin
Keyword(s):  
Arabidopsis Thaliana ◽  
Agrobacterium Tumefaciens ◽  
Transformation Process ◽  
Stable Transformation ◽  
Wild Type ◽  
Dna Integration ◽  
Agrobacterium Tumefaciens Transformation ◽  
Inoculation Assay

The Arabidopsis thaliana mutants uvh1 and rad5, originally identified as radiation hypersensitive, were reported to be deficient in T-DNA integration based on the relative efficiencies of stable transformation and T-DNA transfer. We reassessed these mutants for susceptibility to transformation by Agrobacterium tumefaciens. The mutant rad5 showed a significant reduction in the efficiency of transient as well as stable transformation, compared with its wild-type progenitor. These data indicate that rad5 is blocked at a step in the transformation process prior to T-DNA integration. We additionally found, using both an in vitro root inoculation and an in vivo flower bolt inoculation assay, that the mutant uvh1 is as susceptible to A. tumefaciens-mediated transformation as is its wild-type progenitor, C10.

Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector

2010 ◽  
Vol 107 (4) ◽  
pp. 717-729 ◽  
Author(s):  
Shuohao Huang ◽  
Yoshinori Kawabe ◽  
Akira Ito ◽  
Masamichi Kamihira
Keyword(s):  
Cre Recombinase ◽  
Retroviral Vector ◽  
Site Specific ◽  
Specific Modification ◽  
Cellular Genome
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