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

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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).

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