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Updated: Dec 23, 2025
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells
Ping Lin1, Shugang Qin2, Qinqin Pu2
1Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58203, USA; Wound Trauma Medical Center, State Key Laboratory of Trauma, Burns and Combined Injury, Daping Hospital, Army Medical University, Chongqing 400042, China.
Abstract:
Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.
Insights
Researchers identified novel anti-CRISPR (acr) genes that regulate Cas13a activity for precise RNA editing. These inhibitors control Cas13a function, enhancing applications in nucleic acid detection and disease diagnosis.
Area of Science:
- Molecular Biology
- CRISPR Technology
- RNA Biology
Background:
- Cas13 enzymes are versatile tools for RNA editing, detection, and diagnostics.
- Uncontrolled Cas13 activity can lead to undesirable effects.
- Anti-CRISPR (acr) genes offer a mechanism to modulate CRISPR-Cas systems.
Purpose of the Study:
- To discover and characterize anti-CRISPR (acr) genes that inhibit Cas13a activity.
- To evaluate the potential of these acr genes for controlling RNA editing and detection applications.
Main Methods:
- Screening of anti-CRISPR (acr) gene candidates from phage and prophage regions.
- Evaluation of acr gene effects on Cas13a function in human cells.
- Mechanistic studies on the binding interactions between acr molecules and Cas13a or its complex.
Main Results:
- Discovery of seven acrVIA genes (acrVIA1-7) that inhibit Cas13a.
- Demonstrated significant attenuation of RNA targeting and editing by Cas13a in human cells.
- Showcased the ability of type VI-A anti-CRISPRs (AcrVIAs) to modulate dCas13a's single-nucleic acid editing capability.
- Elucidated specific binding mechanisms of different AcrVIA variants to LwaCas13a or its crRNA complex.
Conclusions:
- The identified acrVIA molecules provide a means to precisely control Cas13a activity.
- These inhibitors hold promise for optimizing Cas13-based RNA editing applications.
- The findings contribute to understanding phage-bacterium interactions and advancing CRISPR technology.
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