Chapter 10
Specific Gene Silencing in Leptospira biflexa by RNA-Guided
Catalytically Inactive Cas9 (dCas9)
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento
Abstract
Easy, practical, and affordable gene silencing techniques are constantly progressing, and genetic tools such
as TALEs, RNAi, and CRISPR/Cas9 have emerged as new techniques for understanding the basic biology
and virulence mechanisms of pathogenic organisms, including bacteria. Here, we describe one-step targeted gene silencing in Leptospira biflexa by using plasmids expressing catalytically inactive Streptococcus
pyogenes Cas9 (dCas9) and a single-guide RNA (sgRNA) capable of pairing to the coding strand of a
desired gene.
Key words Single-guide RNA, dCas9, Gene silencing, Leptospira, Electroporation
1 Introduction
The development of gene inactivation or silencing techniques is a
pivotal step for understanding the basic biology and virulence of
pathogenic microorganisms. In Leptospira spp., these aspects have
remained unexplored mainly due to the lack of genetic tools for
easy and efficient genetic manipulation in spirochetes. Distinct
types of CRISPR (clustered regularly interspaced short palindromic
repeat)/Cas systems have been found in the genome of most
Archaea and Bacteria, playing an important role in their immunity
against phages and plasmids [1, 2]. The type II CRISPR/Cas
system of Streptococcus pyogenes has been widely used in several
cell types for mutagenesis [3–7]. The enzyme Cas9 is the
RNA-guided DNA endonuclease capable of recognizing genomic
sites and causing double-strand breaks (DSB).
Eukaryotic cells can repair DSBs introduced by Cas9 by directly
ligating DNA broken ends in the absence of a repair template
[8, 9]. However, Cas9-induced DSB in the chromosome of most
prokaryotes, including L. biflexa, is lethal to the cells in the absence
of a template for recombination [10–12].
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_10, © Springer Science+Business Media, LLC, part of Springer Nature 2020
109
Specific Gene Silencing in Leptospira biflexa by RNA-Guided
Catalytically Inactive Cas9 (dCas9)
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento
Abstract
Easy, practical, and affordable gene silencing techniques are constantly progressing, and genetic tools such
as TALEs, RNAi, and CRISPR/Cas9 have emerged as new techniques for understanding the basic biology
and virulence mechanisms of pathogenic organisms, including bacteria. Here, we describe one-step targeted gene silencing in Leptospira biflexa by using plasmids expressing catalytically inactive Streptococcus
pyogenes Cas9 (dCas9) and a single-guide RNA (sgRNA) capable of pairing to the coding strand of a
desired gene.
Key words Single-guide RNA, dCas9, Gene silencing, Leptospira, Electroporation
1 Introduction
The development of gene inactivation or silencing techniques is a
pivotal step for understanding the basic biology and virulence of
pathogenic microorganisms. In Leptospira spp., these aspects have
remained unexplored mainly due to the lack of genetic tools for
easy and efficient genetic manipulation in spirochetes. Distinct
types of CRISPR (clustered regularly interspaced short palindromic
repeat)/Cas systems have been found in the genome of most
Archaea and Bacteria, playing an important role in their immunity
against phages and plasmids [1, 2]. The type II CRISPR/Cas
system of Streptococcus pyogenes has been widely used in several
cell types for mutagenesis [3–7]. The enzyme Cas9 is the
RNA-guided DNA endonuclease capable of recognizing genomic
sites and causing double-strand breaks (DSB).
Eukaryotic cells can repair DSBs introduced by Cas9 by directly
ligating DNA broken ends in the absence of a repair template
[8, 9]. However, Cas9-induced DSB in the chromosome of most
prokaryotes, including L. biflexa, is lethal to the cells in the absence
of a template for recombination [10–12].
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_10, © Springer Science+Business Media, LLC, part of Springer Nature 2020
109