CRISPR-mediated Gene silencing with dCas9 or CRISPRi (Part 52- CRISPR in gene editing and beyond)
Welcome to the 52nd part of the multi-part series on applications of CRISPR in gene editing and beyond.
In addition to being fused to transcriptional activators as discussed in Part 51, dCas9 can also function as a repressor. Gene silencing by dCas9 is called CRISPR interference or written as CRISPRi.
When the gRNA-dCas9 complex binds to the region downstream of the promoter or exon sequence of the target DNA, it interferes with transcription by preventing transcriptional factors and RNA polymerase from accessing the promoter of the target DNA, resulting in reversible gene silencing (Qi et al., 2013; Gilbert et al., 2014). However, the efficiency of gene repression by dCas9 alone is relatively low, possibly because it is insufficient to completely disrupt the action of eukaryotic RNA polymerases.
One strategy to improve the efficiency of gene repression is by fusing the dCas9 to the transcriptional repressor KRAB. KRAB stands for Krüppel-associated box, which is the transcriptional repressor domain of the zinc finger proteins such as Kox1 or ZFN10 (Alerasool et al., 2020; Stoll et al., 2022). dCas9-KRAB-gRNA system can achieve the repression level of a targeted gene up to 70–90%.
An all-in-one vector is typically used for the expression of the CRISPRi system. This vector contains both gRNA cloning sites under the U6 promoter and a CMV promoter-driven dCas9-KRAB expression cassette. If multiple genes are required to be targeted, then multiple gRNAs are cloned in the vector. After the expression, dCas9 and KRAB are targeted to the same target gene defined by the gRNA sequence, resulting in its silencing.
Other transcriptional repressors that can be fused to dCas9 are SALL1 and SDS3. CRISPRi has been used to treat familial hypercholesterolemia and obesity in mouse models in vivo by repressing the expression of genes that are involved in the disease pathology.
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References
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173–1183.
Gilbert, L. A., Horlbeck, M. A., Adamson, B., Villalta, J. E., Chen, Y., Whitehead, E. H., … & Weissman, J. S. (2014). Genome-scale CRISPR-mediated control of gene repression and activation. Cell, 159(3), 647–661.
Alerasool, N., Segal, D., Lee, H., & Taipale, M. (2020). An efficient KRAB domain for CRISPRi applications in human cells. Nature Methods, 17(11), 1093–1096.
Stoll, G. A., Pandiloski, N., Douse, C. H., & Modis, Y. (2022). Structure and functional mapping of the KRAB‐KAP1 repressor complex. The EMBO Journal, 41(24), e111179.
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