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RNAi-mediated control of CRISPR functions.

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.

SUBMITTER: Huang X

PROVIDER: S-EPMC7295050 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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RNAi-mediated control of CRISPR functions.

Huang Xinbo X Chen Zhicong Z Liu Yuchen Y

Theranostics 20200517 15

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. <b>Methods</b>: 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 ...[more]

PMID: 32550896

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Project description:Anopheles mosquitoes are the primary vectors for the transmission of malaria parasites, which poses a devastating burden on global public health and welfare. The recent invasion of Anopheles stephensi in Africa has made malaria eradication more challenging due to its outdoor biting behavior and widespread resistance to insecticides. To address this issue, we developed a new approach for mosquito larvae control using gut microbiota-mediated RNA interference (RNAi). We engineered a mosquito symbiotic gut bacterium, Serratia fonticola, by deleting its RNase III gene to produce double-stranded RNAs (dsRNAs) in the mosquito larval gut. We found that the engineered S. fonticola strains can stably colonize mosquito larval guts and produce dsRNAs dsMet or dsEcR to activate RNAi and effectively suppress the expression of methoprene-tolerant gene Met and ecdysone receptor gene EcR, which encode receptors for juvenile hormone and ecdysone pathways in mosquitoes, respectively. Importantly, the engineered S. fonticola strains markedly inhibit the development of A. stephensi larvae and leads to a high mortality, providing an effective dsRNA delivery system for silencing genes in insects and a novel RNAi-mediated pest control strategy. Collectively, our symbiont-mediated RNAi (smRNAi) approach offers an innovative and sustainable method for controlling mosquito larvae and provides a promising strategy for combating malaria. IMPORTANCE Mosquitoes are vectors for various diseases, imposing a significant threat to public health globally. The recent invasion of A. stephensi in Africa has made malaria eradication more challenging due to its outdoor biting behavior and widespread resistance to insecticides. RNA interference (RNAi) is a promising approach that uses dsRNA to silence specific genes in pests. This study presents the use of a gut symbiotic bacterium, Serratia fonticola, as an efficient delivery system of dsRNA for RNAi-mediated pest control. The knockout of RNase III, a dsRNA-specific endonuclease gene, in S. fonticola using CRISPR-Cas9 led to efficient dsRNA production. Engineered strains of S. fonticola can colonize the mosquito larval gut and effectively suppress the expression of two critical genes, Met and EcR, which inhibit mosquito development and cause high mortality in mosquito larvae. This study highlights the potential of exploring the mosquito microbiota as a source of dsRNA for RNAi-based pest control.

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RNAi-mediated control of CRISPR functions.

Publications

RNAi-mediated control of CRISPR functions.

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