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. 2018 Oct 12;362(6411):236-239.
doi: 10.1126/science.aau5138. Epub 2018 Sep 6.

Systematic Discovery of Natural CRISPR-Cas12a Inhibitors

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Free PMC article

Systematic Discovery of Natural CRISPR-Cas12a Inhibitors

Kyle E Watters et al. Science. .
Free PMC article

Abstract

Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. Although bacteria harboring CRISPR-Cas9 or CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we use a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a-mediated genome editing in human cells. We also find a widespread connection between CRISPR self-targeting and inhibitor prevalence in prokaryotic genomes, suggesting a straightforward path to the discovery of many more anti-CRISPRs from the microbial world.

Conflict of interest statement

Competing interests: J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, and Mammoth Biosciences, a scientific adviser to Caribou, Intellia, Scribe, Synthego, Metagenomi, Inari, and eFFECTOR Therapeutics, and a director of Driver and Johnson & Johnson. The Regents of the University of California have patents pending for CRISPR related technologies on which the authors are inventors.

Figures

Fig. 1.
Fig. 1.. Bioinformatic approach for discovering Acr genes.
(A) Anti-CRISPRs allow survival of cells containing self-targeting CRISPR arrays. (B) STSS finds self-targeting CRISPR spacers in genomic DNA, predicts the type of CRISPR system involved, and obtains information about the targeted sequence. (C) A large percentage of genomes containing self-targeting (ST) spacers predicted to be lethal contain previously-identified Acr genes. (D) Moraxella bovoculi strain 22581 contains three self-targeting spacers in two different prophages in the genome. All of the protospacers contain a TTV protospacer-adjacent motif (PAM) (22).
Fig. 2.
Fig. 2.. TXTL screening for Acr gene candidates.
(A) Overview of the transcription-translation (TXTL) reaction. DNA expressing Cas12a, two fluorescent reporters, and two gRNAs are mixed with or without DNA potentially containing Acr genes. (B) Cleavage of the reporter plasmid results in a reduced fluorescent output that is rescued by Acr genes (Acr-absent data in triplicate). (C) Amount of relative inhibition observed for 67 genomic fragments (GFs) across three self-targeting M. bovoculi strains. Four GFs (bold) exhibited inhibition in both fluorescence channels. (D) Genomic fragments GF29, GF35, GF36, and GF59 (99% nucleotide identity to GF29) exhibited high levels of expression for both reporters. (E) Testing the individual genes from the fragments in (D) (table S2) resulted in the identification AcrVA1 (GF36 candidate 1), AcrVA4 (GF59 candidate 2), and AcrVA5 (GF59 candidate 3). (F) Kinetic TXTL data for the AcrVA genes measured over the course of 10 hours of gene expression.
Fig. 3.
Fig. 3.. Biochemical validation of AcrVA inhibitors.
(A) Moraxella bovoculi Cas12a (MbCas12a) in vitro dsDNA cleavage is inhibited by increasing concentrations of AcrVA1, AcrVA4, and AcrVA5 (0 – 1.25 μM; see Methods). (B) LbCas12a, a Cas12a commonly used for gene editing and diagnostics (4, 22, 26), is also inhibited by all three AcrVA proteins. (C) High concentrations of AcrVA1 inhibit AsCas12a-mediated dsDNA cleavage, but AcrVA4 and AcrVA5 have no effect. Triangles indicate uncleaved (black) or cleaved (gray) DNA.
Fig. 4.
Fig. 4.. AcrVAs robustly inhibit genome editing by specific CRISPR-Cas12a nucleases in mammalian cells.
(A) Overview of the editing reporter assay in human cells. (B) Quantification of genome editing in reporter cell lines stably expressing the indicated CRISPR-Cas12a inhibitors (AcrVAs) or a control (mTagBFP2, mCherry). The scale of each plot is adjusted to compensate for differences in editing efficiency. Error bars indicate standard deviations of triplicates. (C) Biochemical analysis of AcrVA-mediated inhibition in representative samples shown in (B). Editing was assessed by the T7 endonuclease 1 (T7E1) assay.

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