Synthetic type III-E CRISPR-Cas effectors for programmable RNA-targeting
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Abstract
The recent discovery of the type III-E CRISPR-Cas effector class has reshaped our fundamental understanding of CRISPR-Cas evolution and classification. Type III-E effectors are composed of several Cas7-like domains and a single Cas11-like domain naturally fused together to create a single polypeptide capable of programmably targeting and degrading RNA. Here we identify a novel composition of a type III-E-like effector composed of Cas7-like and a Cas1-like domain, that can be engineered into an active chimeric RNA-targeting Cas effector and presents a new function of Cas1 in RNA-targeting. Furthermore, we demonstrate a unique modularity of type III-E effectors by methodically substituting domains between orthogonal type III-E proteins to engineer compact synthetic Cas effectors. We refine our methods to generate several compact effectors for programmable RNA-targeting in mammalian cells. Cas7-S represents a new understanding of type III-E architecture and modularity, and provides a platform for engineering genome engineering technologies from the blueprint of nature.
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Protocol*
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Here we demonstrate that the Cas7-11 proteins are modular and can be engineered into compact, efficient, and highly specific Cas7-S effectors for RNA-targeting applications.
Did you explore other offtarget effects?
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We also found Cas7-1 contains a conserved catalytic residue that aligns to all known Cas7.3 domains, as well as zinc-finger motifs unique to type III-E effectors (4) (Figure 1B-E). Lastly, the Cas1 domain aligns to a Reverse Transcriptase-Cas1 (RT-Cas1) fusion protein commonly associated with type III systems of all characterized subtypes (III-A, III-B, III-C, and III-D) (Figure 1F, File S1).
Excellent paper. Why do you think Cas7-1 is missing the highly conserved zinc finger residues in the 499-518 region (Fig 1C)? It seems that the loss of these residues also evolved in the CsbCas7-11 system as well.
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