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Levels of sgRNA as a Major Factor Affecting CRISPRi Knockdown Efficiency in K562 Cells

  • MOLECULAR CELL BIOLOGY
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Abstract

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.

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ACKNOWLEDGMENTS

We thank Professor Jonathan S. Weissman, David E. Root and John G. Doench for sharing their CRISPRi library on Addgene, and we acknowledge Dr. Luke A. Gilbert for offering advice on CRISPRi library.

Funding

This work was supported by the National Natural Science Foundation of China (81860652).

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Authors and Affiliations

  1. College of Life Sciences, Inner Mongolia Agricultural University, 010010, Inner Mongolia, China

    Y. Wang, Z. C. Dong, X. J. Jiang, P. Gong, J. Lu & F. Wan

  2. College of Science, Inner Mongolia Agricultural University, 010010, Inner Mongolia, China

    Y. Xie

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  1. Y. Wang
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  2. Y. Xie
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  3. Z. C. Dong
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  7. F. Wan
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Correspondence to F. Wan.

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This article does not contain any studies involving animals or human participants performed by any of the authors.

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Wang, Y., Xie, Y., Dong, Z.C. et al. Levels of sgRNA as a Major Factor Affecting CRISPRi Knockdown Efficiency in K562 Cells. Mol Biol 55, 75–82 (2021). https://doi.org/10.1134/S0026893321010143

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  • DOI: https://doi.org/10.1134/S0026893321010143

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