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. 2020 May 17;10(15):6661-6673.
doi: 10.7150/thno.44880. eCollection 2020.

RNAi-mediated control of CRISPR functions

Xinbo Huang  1   2   3 , Zhicong Chen  1   2   3 , Yuchen Liu  1   2   3
Affiliations

RNAi-mediated control of CRISPR functions

Xinbo Huang et al. Theranostics. .

Abstract

CRISPR-Cas9 has become a versatile tool for genome editing and regulation, and strategies to effectively control its activity have attracted much attention. RNAi, also a gene-regulating tool, is used as another mechanism by which eukaryotes resist the invasion of foreign genetic material. Methods: In this study, we analyzed the quantitative inhibition of the CRISPR system by using artificial miRNAs (amiRNAs) combined with the RNAi enhancer enoxacin to improve the targeting specificity of the CRISPR system. Furthermore, we examined the feasibility of improving the efficiency of gene editing and regulation by blocking the effects of natural intracellular miRNAs on sgRNAs. Results: amiRNAs targeting the sgRNA were used to control its expression, and the small molecule drug denoxacin was utilized to enhance this effect, especially in the presence of Cas9. amiRNA/enoxacin inhibited CRISPR-mediated gene editing and regulation both in vitro and in vivo and could tune sgRNA-targeting specificity. Furthermore, CRISPR efficiency was increased by blocking the effects of endogenous miRNAs. Conclusion: Our study provides an efficient molecular switch for conditional regulation of CRISPR activities in mammalian cells and also presents potentially useful approaches for solving current key issues of off-target effects and low targeting efficiency.

Keywords: CRISPR switch; artificial miRNA; enoxacin; miRNA sponge.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Effects of amiRNAs on the expression of sgRNAs. (A) Binding sites of amiRNAs in different regions of sgRNA. Region 1 represents the spacer sequence, and regions 2 and 3 are located in the sgRNA backbone. (B) Effects of amiRNAs on the expression of naked sgRNA by qRT-PCR. GAPDH was used as a control. Data are the mean ± SD from five experiments. **P < 0.01, compared with the amiRNA negative control using the paired, one-sided t-test. (C) Effects of amiRNAs on the expression of sgRNA protected by Cas9 protein. GAPDH was used as a control. Data are the mean ± SD from five experiments. *P < 0.05, compared with the amiRNA negative control using the paired, one-sided t-test. (d) Effects of amiRNAs on Cas9-mediated DNMT1 cleavage efficiency. Data are the mean ± SD from five experiments.
Figure 2
Figure 2
Enoxacin promotes amiRNA-mediated CRISPR inhibition. (A) Effects of amiRNAs on the expression of sgRNA at different enoxacin concentrations. amiRNA-NC, negative control amiRNA designed with no known RNA target in cells. sgRNA-NC, negative control sgRNA designed with no target gene in the human genome. Data are the mean ± SD from five experiments. **P < 0.01, compared with the negative control using the paired, one-sided t-test. *P < 0.05, compared with the negative control using the paired, one-sided t-test. (B) Effects of amiRNAs on Cas9-mediated DNMT1 cleavage efficiency at different concentrations of enoxacin. Data are the mean ± SD from five experiments. **P < 0.01, compared with the amiRNA negative control using the paired, one-sided t-test. *P < 0.05, compared with the amiRNA negative control using the paired, one-sided t-test. (C) Effects of amiRNAs on dCas9-mediated DNMT1 transcriptional suppression at different enoxacin concentrations. Data are the mean ± SD from five experiments. **P < 0.01, compared with the amiRNA negative control using the paired, one-sided t-test.
Figure 3
Figure 3
Differential amiRNA-mediated CRISPR inhibition patterns. (A) Effects of amiRNAs with full sgRNA complementarity on Cas9-mediated gene cleavage efficiency. Data are expressed as the mean ± SD. **P < 0.01, compared with the negative control, determined with a paired, one-sided t-test. (B) Effects of amiRNAs with seed site complementarity on Cas9-mediated gene cleavage efficiency. Data are the mean ± SD from five experiments. **P < 0.01, compared with the negative control, determined with a paired, one-sided t-test.
Figure 4
Figure 4
In vivo gene editing and regulation controlled by amiRNAs. (A) AAV expressing the amiRNA or CRISPR system was injected via the tail vein of the mouse. (B) Effects of amiRNAs with full sgRNA complementarity on Cas9-mediated gene cleavage efficiency. Data are the mean ± SD from five experiments. (C) Effects of amiRNAs with full sgRNA complementarity on Cas9-mediated gene inhibitory efficiency. The Apoa1 mRNA level was determined by qRT-PCR. Data are the mean ± SD from five experiments. (D) Effects of amiRNAs with full sgRNA complementarity on Cas9-mediated gene activation efficiency. The Apoa1 mRNA level was determined by qRT-PCR. Data are the mean ± SD from five experiments.
Figure 5
Figure 5
Reduction of off-target events mediated by amiRNA. (A) Effects of amiRNAs with full sgRNA complementarity on sgRNAs. Data are the mean ± SD from five experiments. **P < 0.01, compared with the negative control, determined with a paired, one-sided t-test. (B) Effects of amiRNAs with seed site complementarity on sgRNA. Data are the mean ± SD from five experiments. **P < 0.01, compared with the negative control, determined with a paired, one-sided t-test.
Figure 6
Figure 6
Enhanced on-target efficiency by miRNA sponges. (A) Binding sequences of native miRNAs at different positions of the sgRNA. (B) Expression of different miRNAs in HEK-293T cells by qRT-PCR. U6 was used as an internal control. (C) Effects of miRNA sponge on Cas9-mediated gene cleavage efficiency. Data are the mean ± SD from five experiments. **P < 0.01, compared with the negative control, determined with a paired, one-sided t-test. (D) Effects of miRNA sponge on dCas9-mediated transcriptional inhibition. Data are the mean ± SD from five experiments. **P < 0.01, compared with the negative control, determined with a paired, one-sided t-test.
Figure 7
Figure 7
Roles of RNAi-mediated CRISPR switches. (A) amiRNAs inhibit the activity of the entire CRISPR system and also inhibit the activity of a specific sgRNA. (B) amiRNAs effectively reduce the off-target effects of Cas9 by targeting sgRNA spacers without affecting the editing efficiency of target genes in the presence of a low concentration of enoxacin. (C) amiRNAs increase the targeting efficiency by eliminating the effects of natural miRNAs with miRNA sponge
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