sgRNA level is a major factor affecting CRISPRi knockdown efficiency in K562 cells

Abstract

Objectives:To determine whether nuclease deactivated Cas9 (dCas9) or sgRNA expression level or both determines the knockdown efficiency of CRISPRi. Results:Cell clones expressing KRAB-dCas9 either under the control of the inducible Tet-on system or SFFV promotor were created by lentiviral transduction, and single clones were selected by fluorescence-activated cell sorting (FACS). Six genes with various expression levels were targeted using lentiviral sgRNA from two libraries, and we found that KRAB-dCas9 must reach a certain threshold for the knockdown. 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 in one cell clone, and the knockdown efficiency correlated well with the sgRNA expressing level, which is controlled by a different multiplicity of infection (MOI). Conclusions:Cell that expresses dCas9 above a certain threshold level can effectively knockdown target gene expression, and the knockdown efficiency correlated well with the sgRNA expression level.