dCas9 (endonuclease-deficient Cas9, with D10A and H841A
mutations
relative to the wild-type sequence of S. pyogenes Cas939 (link)) with an N-terminal SV40 nuclear
localization signal (NLS) was codon-optimized for expression in S. cerevisiae and cloned into a pRS314 backbone under
control of the pTPGI promoter.23 (link) The RNA-guided
transcription factors (crisprTFs) were built by fusing four repeats
of the minimal domain of the herpes simplex viral protein 16 (VP16)
to the C-terminus of dCas9 (dCas9_VP64). The crisprTF-expressing plasmid
was then integrated into the TRP1 locus of S. cerevisiae W303.
The reporter plasmids were built by cloning yeast-enhanced gfp under the control of the wild-type or modified pCYC1m
promoter into pRS406 using one-step Gibson assembly. The reporters
for the multiple-gRNA-binding-site experiment (Figure4 A) were built by cloning the corresponding number of binding
sites upstream of the pCYC1m promoter driving production of EBFP2. All reporters were integrated into the bla1 locus of the integrated crisprTF plasmid.
To build gRNA-expressing
plasmids, empty gRNA expressing vectors
were first made by cloning the pRPR1 promoter (an RNA-polymerase-III-dependent
promoter44 (link)), the gRNA handle (flanked by HindIII and Xho1 sites), and the RPR terminator into the
SacI and KpnI sites of either the pRS423 or pRS425
plasmid using one-step Gibson assembly. The specificity determinant
sequence (SDS) for each gRNA was then cloned into the HindIII site of these vectors by one-step Gibson assembly. Sequences
of the constructs used in this study are listed inTable S1, Supporting Information .
mutations
relative to the wild-type sequence of S. pyogenes Cas939 (link)) with an N-terminal SV40 nuclear
localization signal (NLS) was codon-optimized for expression in S. cerevisiae and cloned into a pRS314 backbone under
control of the pTPGI promoter.23 (link) The RNA-guided
transcription factors (crisprTFs) were built by fusing four repeats
of the minimal domain of the herpes simplex viral protein 16 (VP16)
to the C-terminus of dCas9 (dCas9_VP64). The crisprTF-expressing plasmid
was then integrated into the TRP1 locus of S. cerevisiae W303.
The reporter plasmids were built by cloning yeast-enhanced gfp under the control of the wild-type or modified pCYC1m
promoter into pRS406 using one-step Gibson assembly. The reporters
for the multiple-gRNA-binding-site experiment (Figure
sites upstream of the pCYC1m promoter driving production of EBFP2. All reporters were integrated into the bla1 locus of the integrated crisprTF plasmid.
To build gRNA-expressing
plasmids, empty gRNA expressing vectors
were first made by cloning the pRPR1 promoter (an RNA-polymerase-III-dependent
promoter44 (link)), the gRNA handle (flanked by HindIII and Xho1 sites), and the RPR terminator into the
SacI and KpnI sites of either the pRS423 or pRS425
plasmid using one-step Gibson assembly. The specificity determinant
sequence (SDS) for each gRNA was then cloned into the HindIII site of these vectors by one-step Gibson assembly. Sequences
of the constructs used in this study are listed in
