Optimized In Vitro CRISPR/Cas9 Gene Editing Tool in the West Nile Virus Mosquito Vector, Culex quinquefasciatus.
- Resource Type
- Article
- Authors
- Torres, Tran Zen B.; Prince, Brian C.; Robison, Alexis; Rückert, Claudia
- Source
- Insects (2075-4450). Sep2022, Vol. 13 Issue 9, pN.PAG-N.PAG. 13p.
- Subject
- *ARBOVIRUS diseases
*WEST Nile virus
*CULEX quinquefasciatus
*GENOME editing
*MOSQUITO vectors
*CRISPRS
- Language
- ISSN
- 2075-4450
The resulting plasmid (pUb-Cas9-EGFP cU6:6-sgRNA) was generated to coexpress Cas9 endonuclease and eGFP via a self-cleaving T2A peptide along with a gene-specific sgRNA that can be easily inserted with BspQI restriction digest cloning using the same steps as the I Drosophila- i optimized plasmid. The pAc-sgRNA-Cas9 plasmid [[36]] was modified to encode for a Cas9 tagged with mCherry to generate the Drosophila-optimized CRISPR/Cas9 construct (pAc-Cas9-mCherry dU6:2-sgRNA). Distinct sgRNAs targeting the same gene will be cloned into these I Culex i -optimized plasmids, one sgRNA in the plasmid expressing eGFP-tagged Cas9 and the other sgRNA in the plasmid expressing mCherry-tagged Cas9. Data Availability Statement The I Culex- i optimized plasmid pUb-Cas9-EGFP cU6:6-sgRNA described here and its new mCherry counterpart pUb-Cas9-mCherry cU6:6-sgRNA are available on Addgene as plasmids #190597 and #190598, respectively. [Extracted from the article]