For each mechanism, orthogonal cgRNA/trigger pairs were designed
using the reaction pathway engineering tools within NUPACK (nupack.org ).31 (link),32 (link) For bacterial studies, a control gRNA or a cgRNA/trigger plasmid
was transformed into a modified E. coli MG1655 strain
expressing genomically incorporated mRFP and sfGFP.4 (link) Strains were grown overnight in EZ-RDM (Teknova) and then
diluted and grown to mid log phase (≈4 h). Cell density was
normalized with fresh medium containing aTc for induction of silencing
dCas9 expression (and IPTG for the bacterial terminator switch experiments
only). Induced cells were grown for 12 h, with end-point fluorescence
measured via flow cytometry. For mammalian studies, a cgRNA expression
plasmid and a trigger expression plasmid were cotransfected with a
plasmid expressing an inducing dCas9-VPR fusion33 (link) and a reporter plasmid containing a gRNA binding site upstream
of a minimal CMV promoter for dTomato expression.34 (link),35 (link) The four plasmids were transiently transfected into HEK 293T cells
with Lipofectamine 3000 and grown for 24 h, with end-point fluorescence
measured via flow cytometry. Data analysis was performed on cells
expressing high levels of both cgRNA and trigger fluorescent protein
transfection controls. No unexpected or unusually high safety hazards
were encountered.
using the reaction pathway engineering tools within NUPACK (
was transformed into a modified E. coli MG1655 strain
expressing genomically incorporated mRFP and sfGFP.4 (link) Strains were grown overnight in EZ-RDM (Teknova) and then
diluted and grown to mid log phase (≈4 h). Cell density was
normalized with fresh medium containing aTc for induction of silencing
dCas9 expression (and IPTG for the bacterial terminator switch experiments
only). Induced cells were grown for 12 h, with end-point fluorescence
measured via flow cytometry. For mammalian studies, a cgRNA expression
plasmid and a trigger expression plasmid were cotransfected with a
plasmid expressing an inducing dCas9-VPR fusion33 (link) and a reporter plasmid containing a gRNA binding site upstream
of a minimal CMV promoter for dTomato expression.34 (link),35 (link) The four plasmids were transiently transfected into HEK 293T cells
with Lipofectamine 3000 and grown for 24 h, with end-point fluorescence
measured via flow cytometry. Data analysis was performed on cells
expressing high levels of both cgRNA and trigger fluorescent protein
transfection controls. No unexpected or unusually high safety hazards
were encountered.
