Wang Y, Xie Y, Dong Z C, Jiang X J, Gong P, Lu J, Wan F
College of Life Sciences, Inner Mongolia Agricultural University, Inner Mongolia, 010010 China.
College of Science, Inner Mongolia Agricultural University, Inner Mongolia, 010010 China.
Mol Biol (Mosk). 2021 Jan-Feb;55(1):86-95. doi: 10.31857/S0026898421010146.
To determine how nuclease deactivated Cas9 (dCas9) or single-guide RNA (sgRNA) expression levels affect the knockdown efficiency of CRISPRi, we created K562 cell clones expressing KRAB-dCas9 protein either with the inducible Tet-on system or with the constitutive SFFV promotor. 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. The expression level of dCas9 protein/sgRNA levels and the knockdown efficiency were determined by flow cytometry. The cell clone with the highest KRAB-dCas9 expression level achieved effective CRISPRi knockdown. The data describing this clone were statistically different from that on other clones, indicating the strong KRAB-dCas9 expression might be a prerequisite for CRISPRi. By adopting different multiplicity of infection (MOI) in lentiviral transduction of this clone, we modified the expression level of sgRNA and found that the knockdown efficiency was neither affected by the target gene expression level nor correlated with KRAB-dCas9 levels, which remained relatively constant across all knockdown experiments (coefficient of variation = 2.2%). As an example, the following levels of the knockdowns: 74.72, 72.28 and 39.08% for mmadhc, rpia and znf148 genes, respectively, were achieved. These knockdown efficiencies correlated well with the respective sgRNA expression levels. Linear regression models built using this data indicate that the knockdown efficiency may be significantly affected by the levels of both KRAB-dCas9 and sgRNA. Notably, the sgRNA levels have greater impact, being a major factor affecting CRISPRi efficiency.
为了确定核酸酶失活的Cas9（dCas9）或单向导RNA（sgRNA）的表达水平如何影响CRISPRi的敲低效率，我们创建了用诱导型Tet-on系统或组成型SFFV启动子表达KRAB-dCas9蛋白的K562细胞克隆。通过荧光激活细胞分选（FACS）选择单克隆进行进一步研究。在四个具有不同KRAB-dCas9表达水平的细胞克隆中，使用来自两个文库的慢病毒sgRNA靶向六个具有不同表达水平的基因。通过流式细胞术测定dCas9蛋白/sgRNA水平的表达水平和敲低效率。具有最高KRAB-dCas9表达水平的细胞克隆实现了有效的CRISPRi敲低。描述该克隆的数据与其他克隆的数据在统计学上不同，表明强KRAB-dCas9表达可能是CRISPRi的先决条件。通过在该克隆的慢病毒转导中采用不同的感染复数（MOI），我们改变了sgRNA的表达水平，发现敲低效率既不受靶基因表达水平的影响，也与KRAB-dCas9水平无关，在所有敲低实验中KRAB-dCas9水平保持相对恒定（变异系数=2.2%）。例如，分别实现了mmadhc、rpia和znf148基因74.72%、72.28%和39.08%的敲低水平。这些敲低效率与各自的sgRNA表达水平密切相关。使用该数据建立的线性回归模型表明，敲低效率可能受到KRAB-dCas9和sgRNA水平的显著影响。值得注意的是，sgRNA水平的影响更大，是影响CRISPRi效率的主要因素。
