Programmable RNA targeting with the single-protein CRISPR effector Cas7-11

Ahsen Özcan, Rohan Krajeski, Eleonora Ioannidi, Brennan Lee, Apolonia Gardner, Kira S. Makarova, Eugene V. Koonin, Omar O. Abudayyeh () and Jonathan S. Gootenberg ()
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Ahsen Özcan: Massachusetts Institute of Technology
Rohan Krajeski: Massachusetts Institute of Technology
Eleonora Ioannidi: Massachusetts Institute of Technology
Brennan Lee: Massachusetts Institute of Technology
Apolonia Gardner: Massachusetts Institute of Technology
Kira S. Makarova: National Institutes of Health
Eugene V. Koonin: National Institutes of Health
Omar O. Abudayyeh: Massachusetts Institute of Technology
Jonathan S. Gootenberg: Massachusetts Institute of Technology

Nature, 2021, vol. 597, issue 7878, 720-725

Abstract: Abstract CRISPR–Cas interference is mediated by Cas effector nucleases that are either components of multisubunit complexes—in class 1 CRISPR–Cas systems—or domains of a single protein—in class 2 systems1–3. Here we show that the subtype III-E effector Cas7-11 is a single-protein effector in the class 1 CRISPR–Cas systems originating from the fusion of a putative Cas11 domain and multiple Cas7 subunits that are derived from subtype III-D. Cas7-11 from Desulfonema ishimotonii (DiCas7-11), when expressed in Escherichia coli, has substantial RNA interference effectivity against mRNAs and bacteriophages. Similar to many class 2 effectors—and unique among class 1 systems—DiCas7-11 processes pre-CRISPR RNA into mature CRISPR RNA (crRNA) and cleaves RNA at positions defined by the target:spacer duplex, without detectable non-specific activity. We engineered Cas7-11 for RNA knockdown and editing in mammalian cells. We show that Cas7-11 has no effects on cell viability, whereas other RNA-targeting tools (such as short hairpin RNAs and Cas13) show substantial cell toxicity4,5. This study illustrates the evolution of a single-protein effector from multisubunit class 1 effector complexes, expanding our understanding of the diversity of CRISPR systems. Cas7-11 provides the basis for new programmable RNA-targeting tools that are free of collateral activity and cell toxicity.

Date: 2021
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DOI: 10.1038/s41586-021-03886-5

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