scholarly journals Evaluation of Gene Knockouts by CRISPR as Potential Targets for the Genetic Engineering of the Mosquito Culex quinquefasciatus

2021 ◽  
Author(s):  
Xuechun Feng ◽  
Lukas Kambic ◽  
Jared H.K. Nishimoto ◽  
Floyd A. Reed ◽  
Jai A. Denton ◽  
...  
2015 ◽  
Vol 1 (3) ◽  
pp. e1500248 ◽  
Author(s):  
Valmik K. Vyas ◽  
M. Inmaculada Barrasa ◽  
Gerald R. Fink

Candida albicansis a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families. We describe aC. albicansCRISPR system that overcomes many of the obstacles to genetic engineering in this organism. The high frequency with which CRISPR-induced mutations can be directed to target genes enables easy isolation of homozygous gene knockouts, even without selection. Moreover, the system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions. This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome ofCandidaand provides a new window into the biology of this pathogen.


2020 ◽  
Author(s):  
Xuechun Feng ◽  
Lukas Kambic ◽  
Jared H.K. Nishimoto ◽  
Floyd A. Reed ◽  
Jai A. Denton ◽  
...  

AbstractCulex mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people, and separately, poses a conservation threat to insular bird species. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations, yet research in Culex has been lagging compared to other disease vectors, with only a few reports testing the functionality of CRISPR in these species. Here we use this tool to disrupt a set of five pigmentation genes in Culex quinquefasciatus that when altered, lead to visible, homozygous-viable phenotypes. We further validate our approach on two distinct strains of Culex quinquefasciatus that are relevant to potential future public health and bird conservation applications. Lastly, we generate a double-mutant line, demonstrating the possibility of combining multiple such mutations in a single individual. Our work provides a platform of five validated loci that could be used for targeted mutagenesis for research in Culex quinquefasciatus aimed at the development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily-scored transgenesis marker.


2021 ◽  
Author(s):  
Meliawati Meliawati ◽  
Christa Teckentrup ◽  
Jochen Schmid

Clustered regularly interspaced short palindromic repeats (CRISPR) system has rapidly advanced genetic engineering research. The system has been applied for different genetic engineering purposes in multiple organisms including the quite rarely explored Paenibacillus polymyxa. Only limited studies on CRISPR-based system have been described for this highly interesting and versatile bacterium. Here, we demonstrated the utilization of a Cas9-based system to realize 32.8 kb deletion of genomic region by using a single targeting sgRNA. Large cluster deletion was successfully performed with remarkable efficiency of 97 %. Furthermore, we also exploited the system for multiplexing by editing of two distantly located genes at once. We investigated double gene knockouts as well as simultaneous gene integrations and reached editing efficiencies of 78 % and 50 %, respectively. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.


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