4 Notes
1. Previous reports suggested CRISPRi is more efficient when
targeting the 5
0 , rather than 3
0 , end of the ORF. We and others
have not observed such position-dependent differences in
CRISPRi efficacy [9, 20, 21].
2. Evidence from a recent genome-wide CRISPRi screen in E. coli
suggests that stronger transcriptional interference is achieved,
on average, by targeting the gene ORF rather than by targeting
the gene promoter [20]. However, this is not a consensus
viewpoint in the field [22].
3. The canonical SpyCas9 has an optimum sgRNA targeting
sequence length of 20 nt. By contrast, unpublished results
from our lab suggest Sth1Cas9 appears to have an optimum
sgRNA length of 21–24 nt.
4. Off-target effects have been reported in bacterial CRISPRi
systems [21]. In the SpydCas9 CRISPRi system used in
E. coli, Cui and colleagues found that sgRNAs with as few as
9 nucleotides of perfect complementarity between the
PAM-proximal region of the sgRNA targeting sequence (i.e.,
the seed sequence) and a PAM-containing off-target site could
mediate off-target gene repression [21].Given the prevalence
of the SpyCas9 PAM (5
0 -NGG-3
0 ) in the bacterial chromosome, off-target gene repression was common enough to warrant consideration in sgRNA design. The authors recommend
checking a selected sgRNA for off-target effects by identifying
perfect matches to the 9 nucleotide sgRNA seed region. If
there are multiple perfect matches to the bacterial chromosome, and these potential off-target sites have a PAM and
target the nontemplate strand within an ORF, or either strand
in a promoter, an alternatives gRNA should be designed. Note
that the more restrictive PAM for Sth1Cas9 means that
off-target effects are likely to be rarer than those observed
with SpyCas9. Potential off-target effects for Sth1 sgRNAs
can be identified as described above.
5. Cui et al. also observed a phenomenon they describe as the
“bad seed” [21]. Using the Spy dCas9 CRISPRi system, “bad
seeds” were defined as specific 5 nucleotide sequences in the
sgRNA seed sequence that result in target-independent fitness
costs. The mechanism responsible for this phenomenon has
not been identified, but the bad-seed effect could be alleviated
by reducing Spy dCas9 expression levels.
6. ATc is light sensitive. Cultures should be kept in the dark with
minimal light exposure. Flasks and plates can be wrapped in
aluminum foil to minimize light exposure.
360
Andrew I. Wong and Jeremy M. Rock
1. Previous reports suggested CRISPRi is more efficient when
targeting the 5
0 , rather than 3
0 , end of the ORF. We and others
have not observed such position-dependent differences in
CRISPRi efficacy [9, 20, 21].
2. Evidence from a recent genome-wide CRISPRi screen in E. coli
suggests that stronger transcriptional interference is achieved,
on average, by targeting the gene ORF rather than by targeting
the gene promoter [20]. However, this is not a consensus
viewpoint in the field [22].
3. The canonical SpyCas9 has an optimum sgRNA targeting
sequence length of 20 nt. By contrast, unpublished results
from our lab suggest Sth1Cas9 appears to have an optimum
sgRNA length of 21–24 nt.
4. Off-target effects have been reported in bacterial CRISPRi
systems [21]. In the SpydCas9 CRISPRi system used in
E. coli, Cui and colleagues found that sgRNAs with as few as
9 nucleotides of perfect complementarity between the
PAM-proximal region of the sgRNA targeting sequence (i.e.,
the seed sequence) and a PAM-containing off-target site could
mediate off-target gene repression [21].Given the prevalence
of the SpyCas9 PAM (5
0 -NGG-3
0 ) in the bacterial chromosome, off-target gene repression was common enough to warrant consideration in sgRNA design. The authors recommend
checking a selected sgRNA for off-target effects by identifying
perfect matches to the 9 nucleotide sgRNA seed region. If
there are multiple perfect matches to the bacterial chromosome, and these potential off-target sites have a PAM and
target the nontemplate strand within an ORF, or either strand
in a promoter, an alternatives gRNA should be designed. Note
that the more restrictive PAM for Sth1Cas9 means that
off-target effects are likely to be rarer than those observed
with SpyCas9. Potential off-target effects for Sth1 sgRNAs
can be identified as described above.
5. Cui et al. also observed a phenomenon they describe as the
“bad seed” [21]. Using the Spy dCas9 CRISPRi system, “bad
seeds” were defined as specific 5 nucleotide sequences in the
sgRNA seed sequence that result in target-independent fitness
costs. The mechanism responsible for this phenomenon has
not been identified, but the bad-seed effect could be alleviated
by reducing Spy dCas9 expression levels.
6. ATc is light sensitive. Cultures should be kept in the dark with
minimal light exposure. Flasks and plates can be wrapped in
aluminum foil to minimize light exposure.
360
Andrew I. Wong and Jeremy M. Rock