CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira
Published: April 30, 2023
Abstract
Source: Fernandes, L. G. V., et al. Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira. J. Vis. Exp. (2021).
This video demonstrates a CRISPR interference-based gene silencing in pathogenic Leptospira. Targeted repression of gene expression using a CRISPR/Cas system that blocks mRNA formation helps identify candidate genes for pathological and pharmacological studies.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Protospacer definition and plasmid construction NOTE: In this section, the first step of selecting appropriate protospacers for constructing the sgRNA and further ligation into pMaOri.dCas9, is described (Figure 1). This protospacer sequence comprises of a 20 nucle…
Representative Results
The sgRNA cassette should be: cccgggGAACAAGAAAGAGTCAGAGAATTATTGAAGAGATACTCTTATACTACCGTCTTTGGAAGAATTTTCGCATGGATTTTAGATTTGCTGGACTGGTTGAAGCGATTTTTTCAAAAAAAATAATCAATTTGTGTCTGAGATTTGAAAACGCTTGTTTGATAGTTTTTTAAGAATTTCTGATGTTTCAATCGTATAGAAATTCTAAATTTAGAAATCATCCTTTACTTTTCTCTAAGACTTATATAACAATCGCTTTAAACTCAAATTATAATCTTTCAGATAAAAAATTATTCAATATTGATTTACAAAAAATTCCTAAGTTCATACCGTGATTTTCNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTA…
Açıklamalar
The authors have nothing to disclose.
Materials
| 0.1 µm pore size mixed cellulose esters membrane | Millipore | VCWP02500 | Filtration for bacterial conjugation |
| 2,6-Diaminopimelic acid (DAP) | Sigma | D1377 | Growth of auxotrophic E. coli β2163 |
| Agar Noble | BD & Company | 214230 | Used for preparation of solid EMJH and HAN plates |
| Bacto Agar | BD & Company | 214010 | Used for preparation of solid LB plates |
| Glass Microanalysis Filter Holder | Millipore | XX1012530 | Filtration for bacterial conjugation |
| LB broth, Miller | BD & Company | 244620 | Lysogenic liquid medium for E. coli culturing |
| Leptospira Enrichment EMJH | BD & Company | 279510 | Supplementation of EMJH media |
| Leptospira Medium Base EMJH | BD & Company | 279410 | EMJH medium for Leptospira |
| Optical density reader | Molecular Devices | SpectraMax M2 | For optical density measurements of bacterial cultures |
| Spectinomycin | Sigma | S0692 | Selection of pMaOri backbone plasmids |
| Thymidine (dT) | Sigma | T9250 | Growth of auxotrophic E. coli π1 |
| XmaI restriction enzyme | New Englan BioLabs | R0180L | Digestion of plasmids and inserts |
Etiketler
Bu Makaleden Alıntı Yapın
CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira. J. Vis. Exp. (Pending Publication), e20975, doi: (2023).