ORF [9, 10], although it can be effective when targeting the
promoter [21].
2. PAM choice: the consensus PAM sequence for Sth1 dCas9 is
5
0 -NNAGAAW-3
0 [13]. Sth1 dCas9 can recognize noncanonical PAMs with mutations within this consensus sequence at the
cost of the magnitude of target gene knockdown [9]. Importantly, this PAM complexity provides a facile method of tuning
target gene knockdown to generate an allelic series. We have
ranked the approximate PAM “strength” based on the magnitude of CRISPRi knockdown of a Renilla luciferase target gene
in M. smegmatis (Table 1). Numerous factors are likely to
impact the efficacy of a given sgRNA (sgRNA target sequence
composition, location of endogenous RNA polymerase pause
sites, etc.), so PAM “strength” should be interpreted as a
prediction but not a rule for CRISPRi knockdown efficiency.
3. To achieve CRISPRi knockdown, identify PAM sequences in
the template strand of your gene of interest (see Notes 1 and
2). Select a PAM with a desired approximate targeting strength
(Table 1).
4. To design the sgRNA oligos, extract the 21–24 nucleotides of
DNA sequence immediately upstream of the PAM (see Note 3)
such that the 5
0 nucleotide of the targeting sequence is an A or
G. This is the first base that will be transcribed to synthesize the
sgRNA, and A or G is strongly preferred as an initiating nucleotide in mycobacteria [23]. If there is no 5
0 A/G in this 21–24
nucleotide window, select 23 bases immediately upstream of
the PAM and append a 5
0 G to the selected sequence. This
21–24 nucleotide sequence will be the sgRNA targeting
sequence (see Notes 4 and 5 regarding off-target effects and
“bad seed” sequences).
5. Append a 5
0 -GGGA-3
0 sequence 5
0 to the sgRNA targeting
sequence and order this as the top oligo for cloning the
sgRNA.
6. Take the reverse complement of the sgRNA targeting sequence
and append a 5
0 -AAAC-3
0 sequence 5
0 to the reverse complemented sgRNA targeting sequence. This is the bottom oligo
for cloning the sgRNA.
7. When annealed, the top and bottom oligos will form a dsDNA
fragment with sticky end overhangs (the bolded sequences in
the examples below) for ligation into the CRISPRi backbone.
As controls, we provide two pairs of oligos for cloning an
sgRNA against rpoB (rv0667) in M. tuberculosis and an
sgRNA against mmpL3 (MSMEG_0250) in M. smegmatis. Successful CRISPRi targeting of rpoB and mmpL3 will result in a
severe growth defect and serve as a positive control for CRISPRi implementation.
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Andrew I. Wong and Jeremy M. Rock
promoter [21].
2. PAM choice: the consensus PAM sequence for Sth1 dCas9 is
5
0 -NNAGAAW-3
0 [13]. Sth1 dCas9 can recognize noncanonical PAMs with mutations within this consensus sequence at the
cost of the magnitude of target gene knockdown [9]. Importantly, this PAM complexity provides a facile method of tuning
target gene knockdown to generate an allelic series. We have
ranked the approximate PAM “strength” based on the magnitude of CRISPRi knockdown of a Renilla luciferase target gene
in M. smegmatis (Table 1). Numerous factors are likely to
impact the efficacy of a given sgRNA (sgRNA target sequence
composition, location of endogenous RNA polymerase pause
sites, etc.), so PAM “strength” should be interpreted as a
prediction but not a rule for CRISPRi knockdown efficiency.
3. To achieve CRISPRi knockdown, identify PAM sequences in
the template strand of your gene of interest (see Notes 1 and
2). Select a PAM with a desired approximate targeting strength
(Table 1).
4. To design the sgRNA oligos, extract the 21–24 nucleotides of
DNA sequence immediately upstream of the PAM (see Note 3)
such that the 5
0 nucleotide of the targeting sequence is an A or
G. This is the first base that will be transcribed to synthesize the
sgRNA, and A or G is strongly preferred as an initiating nucleotide in mycobacteria [23]. If there is no 5
0 A/G in this 21–24
nucleotide window, select 23 bases immediately upstream of
the PAM and append a 5
0 G to the selected sequence. This
21–24 nucleotide sequence will be the sgRNA targeting
sequence (see Notes 4 and 5 regarding off-target effects and
“bad seed” sequences).
5. Append a 5
0 -GGGA-3
0 sequence 5
0 to the sgRNA targeting
sequence and order this as the top oligo for cloning the
sgRNA.
6. Take the reverse complement of the sgRNA targeting sequence
and append a 5
0 -AAAC-3
0 sequence 5
0 to the reverse complemented sgRNA targeting sequence. This is the bottom oligo
for cloning the sgRNA.
7. When annealed, the top and bottom oligos will form a dsDNA
fragment with sticky end overhangs (the bolded sequences in
the examples below) for ligation into the CRISPRi backbone.
As controls, we provide two pairs of oligos for cloning an
sgRNA against rpoB (rv0667) in M. tuberculosis and an
sgRNA against mmpL3 (MSMEG_0250) in M. smegmatis. Successful CRISPRi targeting of rpoB and mmpL3 will result in a
severe growth defect and serve as a positive control for CRISPRi implementation.
350
Andrew I. Wong and Jeremy M. Rock