Abstract
To determine whether nuclease deactivated Cas9 (dCas9) or sgRNA expression level or both determine the knockdown efficiency of CRISPRi, we carried out the following experiments: Cell clones expressing KRAB-dCas9 either under the control of the inducible Tet-on system or SFFV promotor were created by lentiviral transduction, and several single clones were selected by fluorescence-activated cell sorting (FACS) for further study. Six genes with various expression levels were targeted using lentiviral sgRNA from two libraries, and the only clone with the high expression level of KRAB-dCas9 effectively knocked down target gene expression. In the high KRAB-dCas9 expressing cell clone, the knockdown efficiency was neither affected by the target gene expression level nor does it correlate with the KRAB-dCas9 expression level, which remained relatively constant (CV=2.2%) across knockdown experiments. 74.72%, 72.28%, 39.08% knockdown of MMADHC, RPIA, ZNF148 genes were achieved, and the knockdown efficiency correlated well with the sgRNA expressing level, which is controlled by a different multiplicity of infection (MOI). Cell clones with low KRAB-dCas9 expression levels did not achieve high knockdown efficiency.