In addition to the complete loss-of-function perturbations made possible by Cas9 nuclease, the development of dCas9-based gene repression has enabled reversible, multiplexed, and loss-of-function studies of genes of interest. CRISPR-dCas9-based gene repression can repress transcription by directly blocking RNA polymerase activity (dCas9) or through effector domain-mediated transcriptional silencing (dCas9-KRAB). Studies demonstrate that this technology is highly specific with minimal off-target effects in bacterial cells and allows for tunable regulation of individual genes and multiplexable control of many genes using multiple sgRNAs. Exploring these unique features, CRISPR regulation has been applied to systematically interrogate the function of essential genes for their roles in growth, death, drug resistance, and morphology control.
In addition to being fused to transcriptional activators, dCas9 can also function as a repressor. Cas9 nuclease can be converted into deactivated Cas9 (dCas9), an RNA-programmable DNA-binding protein, by mutating two key residues within its nuclease domains. The use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. Altogether, the results demonstrate that directing dCas9 to different gene regions can prevent both the initiation and elongation of transcription. Interestingly, dCas9 targeting of the coding strand blocks transcription more efﬁciently than targeting of the non-coding strand, suggesting a more efﬁcient displacement of this protein by the elongating RNAP in this conﬁguration.
This was ﬁrst demonstrated in bacteria, where dCas9 alone was able to repress transcription. As CRISPR-dCas9 interferes with the transcription of the targeted genes by sterically hindering the elongation of RNAP or inhibiting the initial binding of RNAP to the promoter, this approach is termed CRISPR interference. This provides a very efﬁcient way to silence transcription in bacteria. In prokaryotes, repression of up to 1000-fold was achieved when targeting dCas9 to either DNA strand within a promoter or to the non-template DNA strand downstream. Repression is tunable, as the choice of sgRNA site determines the strength of its repressive effect. This system is advantageous because genes can be efﬁciently repressed without the addition of speciﬁc effectors, making the repression system simpler and more transferable across genes, species, and cell types than the activation system.
Fig 1. Various engineered CRISPR repression systems.
Although CRISPR-dCas9 mediated gene repression using only dCas9 protein and an sgRNA is highly efficient and can silence gene expression by up to 99.9% in prokaryotes. In eukaryotic cells such steric repression is weaker, it achieves only modest repression (up to 60-80%) of fluorescent reporter genes or tested endogenous genes in mammalian cells. It is possible that the dCas9-sgRNA complex alone is not sufficient to fully block the action of the RNA polymerase (RNAP) complex in eukaryotic cells. As a notable exception, synthetic promoters speciﬁcally constructed for direct repression by dCas9 can be repressed up to 100-fold in mammalian cells. More detailed researches are needed in the future.
To improve the efﬁciency of repression in mammalian cells, dCas9 has been fused with a number of repressive transcriptional or epigenetic effector domains, including the KRAB (Krüppel-associated box) domain of Kox1, the CS (chromoshadow) domain of HP1α, the WPRW domain of Hes1, or four concatenated copies of the mSin3 interaction domain (SID4X). Of these, the KRAB-dCas9 fusion mediated transcriptional repression is proven to be relatively strong and highly speciﬁc in both yeast and mammalian cells, which can lead to downregulation of the endogenous gene in the range of 90 to 99% with a properly designed sgRNA. While these strategies have proven quite effective at repressing transcription, there are improvements that can be made. For example, we have found that using an N-terminal KRAB fusion is more effective at repression than using a C-terminal fusion.
CRISPR-dCas9-based gene repression also offers several advantages over previous forms of gene repression, although it is not necessarily the best choice for every assay. For example, techniques based on RNA interference (RNAi), which consists of small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), allow for sequence-specific repression of endogenous genes of interest in eukaryotic organisms. This gene silencing effect is mediated by transcript-specific degradation due to Watson-Crick base-pairing between mRNA and siRNA or shRNA. The pathways are naturally occurring cellular processes used to regulate mRNA translation and stability. Compared to these targeting techniques, CRISPR-dCas9-based gene repression systems are easier to design, highly specific and efficient, cost effective, and well-suited for high-throughput and multiplexed gene repression across many cell types and organisms.
|Repression method||Engineering||Cost||Ability to target non-coding RNAs||Ability to target small RNAs||Used in pooled genome wide screens||Off-target effects||Limitations in use|
|CRISPR repression||Simple||Low||Yes||Yes||Yes||Minimal||Requries an "NGG" PAM|
|TALE or ZFN||Challenging||High||Yes||Yes||No||Minimal||ZFN requires some engineering to target a sequence|
Ultimately, the choice of whether to use CRISPRi or RNAi will depend on the requirements of a given user. For small-scale use targeting only a few genes, CRISPRi has simpler design rules can achieve very high levels of knockdown. However, RNAi can be advantageous in that one can target speciﬁc splice variants over others, which is not possible with CRISPRi unless the different variants have different transcription start sites. Additionally, it has been shown that off-target effects from siRNAs can result in cell toxicity in a cell type-dependent manner. This has not yet been seen with CRISPRi, although the possibility has not been systematically investigated. It may be that certain cell types tolerate RNAi or CRISPRi better. Finally, CRISPRi is a two-component system, whereas RNAi is a one-component system. In assays where delivering two components may be an issue, it may be more desirable to use RNAi.
In the study of noncoding RNAs (ncRNAs), CRISPR-dCas9-based gene repression also offers many advantages. Noncoding RNAs such as miRNAs and lncRNAs can be targeted by CRISPRi in the same manner as coding genes. Since many miRNAs are redundant, one can potentially make use of the multiplexing capability of CRISPRi to hit all miRNAs in the same targeting "family" at once. One alternative approach to CRISPRi is the use of antagomir miRNA inhibitors, which are modiﬁed oligonucleotides that are antisense with respect to the target miRNA. They bind to the miRNA with high afﬁnity and prevent it from acting on its target mRNA. However, these miRNA inhibitors can be expensive and are speciﬁc for a single miRNA.
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