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

Abstract

To determine how nuclease deactivated Cas9 (dCas9) or sgRNA expression level affects the knockdown efficiency of CRISPRi, K562 cell clones expressing KRAB-dCas9 protein either with the inducible Tet-on system or with the constitutive SFFV promotor were created by lentiviral transduction, and 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 in four cell clones with various KRAB-dCas9 expression levels. We determined the knockdown efficiency and the expression level of the dCas9 protein /sgRNA level by flow cytometry. The cell clone with the highest KRAB-dCas9 expression level achieved effective CRISPRi knockdown, and is statistically different from other clones, indicating enough KRAB-dCas9 expression might be a prerequisite for CRISPRi. Utilizing this clone, we modified the expression level of sgRNA by adopting different multiplicity of infection (MOI)in lentiviral transduction and found that the knockdown efficiency was neither affected by the target gene expression level nor does it correlate with KRAB-dCas9 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 expression level. Linear regression modeling of the data revealed that the knockdown efficiency is significantly affected by both KRAB-dCas9 and sgRNA level, and the sgRNA level has a greater impact, based on the standardized coefficient (0.525 for KRAB-dCas9, 0.981 for sgRNA), indicating that sgRNA level is a major factor affecting CRISPRi efficiency.