CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira

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 nucleotides sequence against the desired target.

1. Obtain the nucleotide sequence of the gene of interest for silencing at GenBank (https://www.ncbi.n....

Results

The sgRNA cassette should be: cccgggGAACAAGAAAGAGTCAGAGAATTATTGAAGAGATACTCTTATACTACCGTCTTTGGAAGAATTTTCGCATGGATTTTAGATTTGCTGGACTGGTTGAAGCGATTTTTTCAAAAAAAATAATCAATTTGTGTCTGAGATTTGAAAACGCTTGTTTGATAGTTTTTTAAGAATTTCTGATGTTTCAATCGTATAGAAATTCTAAATTTAGAAATCATCCTTTACTTTTCTCTAAGACTTATATAACAATCGCTTTAAACTCAAATTATAATCTTTCAGATAAAAAATTATTCAATATTGATTTACAAAAAATTCCTAAGTTCATACCGTGATTTTCNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTA.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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