19. Chemiluminescent substrate.
20. Imager system set for chemiluminescence.
3 Methods
3.1 Selection
of Protospacer
and Plasmid Ligation
In this section, we describe the steps for selecting appropriate
protospacers for constructing and expressing sgRNA and further
ligation into pMaOri.dCas9. This sgRNA will be recognized by the
expressed dCas9 and will direct this protein to genomic targets
based on Watson-Crick base pairing. This complex, bound to the
coding strand of the desired gene, will hamper RNA polymerase
elongation and, therefore, block transcription (Fig. 1). Both dCas9
and sgRNA are expressed in the same plasmids, allowing one-step
gene silencing in L. biflexa. All cloning procedures are performed in
the dT auxotrophic E. coli strain π1 [14], due to pMaOri [15]
origin of replication, R6K-gamma (Fig. 1)
1. The backbone of sgRNA cassette ligation is the plasmid
pMaOri.dCas9 [12] (Fig. 1). This plasmid allows transformation of Leptospira spp. by electroporation and conjugation
[15]. In our experience, the XmaI site has been successfully
used for inclusion of the sgRNA sequence.
2. Select the coding sequence of the desired gene (https://www.
ncbi.nlm.nih.gov/genbank) and submit it to the CHOPCHOP webserver (http://chopchop.cbu.uib.no/), selecting
“Fasta Target” and setting to “CRISPR/Cas9.” Cas9 from
S. pyogenes recognizes the PAM (protospacer adjacent motif)
NGG. The NGG trinucleotide must not be included in the
sgRNA sequence.
3. Based on the output, select the protospacers with the best score
(green arrow) and as close as possible to the 5
0 end of the coding
region. It is very important to select a protospacer that is
capable of pairing to the coding strand. In the output, these
protospacers will be listed as minus strand. We recommend
selecting also a protospacer to hybridize to the template strand,
to serve as control. Around 30–50% gene silencing is expected
in this situation, in contrast to 100% gene silencing when the
sgRNA contains protospacer sequence pairing to the coding
strand (see Note 3).
4. The 20 nt sequence selected referring to the protospacers
should be fused to the lipL32 promoter (at its 5
0 end) and
sgRNA scaffold (3
0 end, including a dCas9 handle and intrinsic
terminator) (Fig. 2). We use the promoter region comprising
À334 to the TSS (transcription start site, according to Zhukova et al. [16]) and scaffold sequence: GTTTTAGAGCTA114
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento
20. Imager system set for chemiluminescence.
3 Methods
3.1 Selection
of Protospacer
and Plasmid Ligation
In this section, we describe the steps for selecting appropriate
protospacers for constructing and expressing sgRNA and further
ligation into pMaOri.dCas9. This sgRNA will be recognized by the
expressed dCas9 and will direct this protein to genomic targets
based on Watson-Crick base pairing. This complex, bound to the
coding strand of the desired gene, will hamper RNA polymerase
elongation and, therefore, block transcription (Fig. 1). Both dCas9
and sgRNA are expressed in the same plasmids, allowing one-step
gene silencing in L. biflexa. All cloning procedures are performed in
the dT auxotrophic E. coli strain π1 [14], due to pMaOri [15]
origin of replication, R6K-gamma (Fig. 1)
1. The backbone of sgRNA cassette ligation is the plasmid
pMaOri.dCas9 [12] (Fig. 1). This plasmid allows transformation of Leptospira spp. by electroporation and conjugation
[15]. In our experience, the XmaI site has been successfully
used for inclusion of the sgRNA sequence.
2. Select the coding sequence of the desired gene (https://www.
ncbi.nlm.nih.gov/genbank) and submit it to the CHOPCHOP webserver (http://chopchop.cbu.uib.no/), selecting
“Fasta Target” and setting to “CRISPR/Cas9.” Cas9 from
S. pyogenes recognizes the PAM (protospacer adjacent motif)
NGG. The NGG trinucleotide must not be included in the
sgRNA sequence.
3. Based on the output, select the protospacers with the best score
(green arrow) and as close as possible to the 5
0 end of the coding
region. It is very important to select a protospacer that is
capable of pairing to the coding strand. In the output, these
protospacers will be listed as minus strand. We recommend
selecting also a protospacer to hybridize to the template strand,
to serve as control. Around 30–50% gene silencing is expected
in this situation, in contrast to 100% gene silencing when the
sgRNA contains protospacer sequence pairing to the coding
strand (see Note 3).
4. The 20 nt sequence selected referring to the protospacers
should be fused to the lipL32 promoter (at its 5
0 end) and
sgRNA scaffold (3
0 end, including a dCas9 handle and intrinsic
terminator) (Fig. 2). We use the promoter region comprising
À334 to the TSS (transcription start site, according to Zhukova et al. [16]) and scaffold sequence: GTTTTAGAGCTA114
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento