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…
Divulgazioni
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
CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira. J. Vis. Exp. (Pending Publication), e20975, doi: (2023).