transcriptional repression. When targeting operonic genes with
CRISPRi, it is therefore important to consider whether the
genes downstream of the targeted gene could be contributing
to or the source of the phenotype of interest. Peters and
colleagues reported polar effects on upstream genes [19],
where genes upstream of the CRISPRi-targeted gene were
also repressed. We and others have not observed this
“upstream” polar effect [9, 21].
13. If not supplied in the expression vector, the promoter driving
expression of the complementation allele can be selected by the
user. In NEBuilder, add the promoter sequence as an additional fragment between the expression vector backbone and 5
0
end of the first gene fragment. PCR amplify the promoter
sequence and clone into the expression vector as per NEBuilder instructions.
14. In order for this vector to be compatible with the CRISPRi
backbone, ensure that the expression vector does not rely on
kanamycin resistance for positive selection or the L5 integrase
for chromosomal integration.
15. The first amplified fragment should contain an upstream overhang with sequence identity to one end of the linearized
expression vector, and a downstream overhang with the silent
mutations and sequence identity to the upstream end of the
second fragment. The second amplified fragment should contain an upstream overhang with the silent mutations and
sequence identity to the downstream end of the previous fragment, and a downstream overhang with sequence identity to
the other end of the linearized expression vector.
16. Lack of complementation by expression of a CRISPRi-sensitive
allele confirms that complementation of the CRISPRi knockdown phenotype by a CRISPRi-resistant allele is not due to the
expression of a second allele of the targeted gene, but rather
due to the specific expression of a CRISPRi-resistant allele of
the targeted gene.
17. The Golden Gate reaction will digest the desired parent sgRNA
plasmid (i.e., the CRISPRi plasmid that contains your first
sgRNA at the BsmBI-based cloning site) and the
PCR-amplified and gel-purified sgRNA cassettes with SapI
and simultaneously ligate with T4 DNA ligase. If there are
difficulties with cloning efficiency using the described Golden
Gate approach, the restriction digests, gel purification, and
ligations can be performed in sequential steps to facilitate
troubleshooting.
18. These thermocycler conditions are suggested for up to
5 inserts. For 6 or more inserts, cycle 30 times between
362
Andrew I. Wong and Jeremy M. Rock
CRISPRi, it is therefore important to consider whether the
genes downstream of the targeted gene could be contributing
to or the source of the phenotype of interest. Peters and
colleagues reported polar effects on upstream genes [19],
where genes upstream of the CRISPRi-targeted gene were
also repressed. We and others have not observed this
“upstream” polar effect [9, 21].
13. If not supplied in the expression vector, the promoter driving
expression of the complementation allele can be selected by the
user. In NEBuilder, add the promoter sequence as an additional fragment between the expression vector backbone and 5
0
end of the first gene fragment. PCR amplify the promoter
sequence and clone into the expression vector as per NEBuilder instructions.
14. In order for this vector to be compatible with the CRISPRi
backbone, ensure that the expression vector does not rely on
kanamycin resistance for positive selection or the L5 integrase
for chromosomal integration.
15. The first amplified fragment should contain an upstream overhang with sequence identity to one end of the linearized
expression vector, and a downstream overhang with the silent
mutations and sequence identity to the upstream end of the
second fragment. The second amplified fragment should contain an upstream overhang with the silent mutations and
sequence identity to the downstream end of the previous fragment, and a downstream overhang with sequence identity to
the other end of the linearized expression vector.
16. Lack of complementation by expression of a CRISPRi-sensitive
allele confirms that complementation of the CRISPRi knockdown phenotype by a CRISPRi-resistant allele is not due to the
expression of a second allele of the targeted gene, but rather
due to the specific expression of a CRISPRi-resistant allele of
the targeted gene.
17. The Golden Gate reaction will digest the desired parent sgRNA
plasmid (i.e., the CRISPRi plasmid that contains your first
sgRNA at the BsmBI-based cloning site) and the
PCR-amplified and gel-purified sgRNA cassettes with SapI
and simultaneously ligate with T4 DNA ligase. If there are
difficulties with cloning efficiency using the described Golden
Gate approach, the restriction digests, gel purification, and
ligations can be performed in sequential steps to facilitate
troubleshooting.
18. These thermocycler conditions are suggested for up to
5 inserts. For 6 or more inserts, cycle 30 times between
362
Andrew I. Wong and Jeremy M. Rock