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

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 systems.sup.1-3. 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 toxicity.sup.4,5. 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. Cas7-11--the fusion of a putative Cas11 domain and four Cas7 subunits--cleaves RNA without detectable non-specific activity and, when optimized for RNA knockdown and editing in mammalian cells, has no effects on cell viability.
Author(s): Ahsen Ã-zcan [sup.1] , Rohan Krajeski [sup.1] , Eleonora Ioannidi [sup.1] [sup.2] , Brennan Lee [sup.1] , Apolonia Gardner [sup.1] [sup.3] , Kira S. Makarova [sup.4] , Eugene V. [...]