Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Molecular Cellular Biology, Harvard University, Cambridge, MA, USA.
⁴ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA.
⁵ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: zhang@broadinstitute.org.

PMID: 39970912
DOI: 10.1016/j.cell.2025.01.034

Free article

Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins

Shai Zilberzwige-Tal et al. Cell. 2025.

Free article

. 2025 Apr 3;188(7):1925-1940.e20.

doi: 10.1016/j.cell.2025.01.034. Epub 2025 Feb 18.

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Molecular Cellular Biology, Harvard University, Cambridge, MA, USA.
⁴ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA.
⁵ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: zhang@broadinstitute.org.

PMID: 39970912
DOI: 10.1016/j.cell.2025.01.034

Abstract

Despite ongoing efforts to study CRISPR systems, the evolutionary origins giving rise to reprogrammable RNA-guided mechanisms remain poorly understood. Here, we describe an integrated sequence/structure evolutionary tracing approach to identify the ancestors of the RNA-targeting CRISPR-Cas13 system. We find that Cas13 likely evolved from AbiF, which is encoded by an abortive infection-linked gene that is stably associated with a conserved non-coding RNA (ncRNA). We further characterize a miniature Cas13, classified here as Cas13e, which serves as an evolutionary intermediate between AbiF and other known Cas13s. Despite this relationship, we show that their functions substantially differ. Whereas Cas13e is an RNA-guided RNA-targeting system, AbiF is a toxin-antitoxin (TA) system with an RNA antitoxin. We solve the structure of AbiF using cryoelectron microscopy (cryo-EM), revealing basic structural alterations that set Cas13s apart from AbiF. Finally, we map the key structural changes that enabled a non-guided TA system to evolve into an RNA-guided CRISPR system.

Keywords: CRISPR; Cas13; RNA-guided mechanism; bacterial immunity; toxin-antitoxin system.

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Conflict of interest statement

Declaration of interests F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, Aera Therapeutics, and Moonwalk Biosciences. F.Z. is a scientific advisor for Octant.

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Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins

Affiliations

Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins

Authors

Affiliations

Abstract

Conflict of interest statement

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