scholarly journals Site-specific Gene Insertion Mediated by a Cre-loxP-carrying Lentiviral Vector

2010 ◽  
Vol 18 (10) ◽  
pp. 1814-1821 ◽  
Author(s):  
Gilles Michel ◽  
Yin Yu ◽  
Tammy Chang ◽  
Jiing-Kuan Yee
Keyword(s):  
Lentiviral Vector ◽  
Specific Gene ◽  
Site Specific ◽  
Gene Insertion

scholarly journals Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

2015 ◽  
Vol 169 (2) ◽  
pp. 931-945 ◽  
Author(s):  
Sergei Svitashev ◽  
Joshua K. Young ◽  
Christine Schwartz ◽  
Huirong Gao ◽  
S. Carl Falco ◽  
...  
Keyword(s):  
Gene Editing ◽  
Targeted Mutagenesis ◽  
Specific Gene ◽  
Site Specific ◽  
Guide Rna ◽  
Gene Insertion

scholarly journals Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

2015 ◽  
Vol 112 (13) ◽  
pp. 4038-4043 ◽  
Author(s):  
Sanjay Basu ◽  
Azadeh Aryan ◽  
Justin M. Overcash ◽  
Glady Hazitha Samuel ◽  
Michelle A. E. Anderson ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Specific Gene ◽  
Gene Drive ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Gene Insertion ◽  
Guide Rnas ◽  
Essential Components ◽  
Drive Systems

Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24–90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2–3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

scholarly journals An Optimized and Efficient CRISPR/Cas9 System for the Endophytic Fungus Pestalotiopsis fici

2021 ◽  
Vol 7 (10) ◽  
pp. 809
Author(s):  
Xinran Xu ◽  
Runye Huang ◽  
Wen-Bing Yin
Keyword(s):  
Natural Products ◽  
Endophytic Fungi ◽  
Endophytic Fungus ◽  
Genetic Manipulation ◽  
Biological Study ◽  
Specific Gene ◽  
Site Specific ◽  
Gene Insertion ◽  
Pestalotiopsis Fici ◽  
Dna Fragment

Endophytic fungi are emerging as attractive producers of natural products with diverse bioactivities and novel structures. However, difficulties in the genetic manipulation of endophytic fungi limit the search of novel secondary metabolites. In this study, we improved the polyethylene glycol (PEG)-mediated protoplast transformation method by introducing the CRISPR/Cas9 system into endophytic fungus Pestalotiopsis fici. Using this approach, we performed genome editing such as site-specific gene insertion, dual-locus mutations, and long DNA fragment deletions in P. fici efficiently. The average efficiency for site-specific gene insertion and two-site gene editing was up to 48.0% and 44.4%, respectively. In addition, the genetic manipulation time with long DNA fragment (5–10 kb) deletion was greatly shortened to one week in comparison with traditional methods such as Agrobacterium tumefaciens-mediated transformation (ATMT). Taken together, the development of the CRISPR/Cas9 system in the endophytic fungus will accelerate the discovery of novel natural products and further biological study.

scholarly journals Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Quang Vinh Phan ◽  
Jörg Contzen ◽  
Petra Seemann ◽  
Manfred Gossen
Keyword(s):  
Chromosomal Gene ◽  
Site Specific ◽  
Flp Recombinase ◽  
Gene Insertion ◽  
Cas9 Nuclease

scholarly journals Site-specific gene expression analysis using an automated tissue micro-dissection punching system

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Takuya Yoda ◽  
Masahito Hosokawa ◽  
Kiyofumi Takahashi ◽  
Chikako Sakanashi ◽  
Haruko Takeyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Specific Gene ◽  
Site Specific ◽  
Specific Gene Expression
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