Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus

Figures

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  Figure 1. The dCmr4α-induced cell death is dependent on the activities of Cmr2α. (A) Deletion of the cmr-α module abolished the Cmr4α-D27A toxicity. pCmr4α and pCmr4α-D27A were transformed into ∆βE233S1 and ∆α∆βE233S1, respectively, and the transformation efficiency was calculated. (B) Cmr4α-D27A toxicity in ∆βE233 and the cmr2α mutant strains. pCmr4α and pCmr4α-D27A were transformed into ΔβE233(WT) and the cmr2α mutants, respectively, and the transformation efficiency was calculated.

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  Figure 2. The Cmr2α-based activities are not equally efficient in mediating the antiviral defense. Self-targeting activity in the cmr2α mutant strains. (A) Outline of designed target sites for testing Cmr-α immunity in S. islandicus. (B) The control plasmid pSeSD1 and the self-targeting plasmids pAC-alba and pAC-RG1 were transformed into ∆βE233(WT) and the cmr2α mutant strains, respectively, and the transformation efficiency was calculated. (C) The control plasmid pSeSD1 and the self-targeting plasmid pS10i were transformed into ∆βE233(WT) and the cmr2α mutant strains, respectively, and the transformation efficiency was calculated. Alba encodes a crenarchaeal chromatin protein; RG1 codes for a reverse gyrase topoisomerase.

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  Figure 3. Contribution of CRISPR array and target sequence to the Cmr4α-D27A toxicity. Transformation efficiency was determined for pCmr4α and pCmr4α-D27A plasmids individually with three different host strains: ∆βE233(WT), ∆CRISPR∆β and ∆CRISPRΔlacS∆β(∆array).

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  Figure 4. Function of Cmr4α conserved amino acids in the Cmr-α immune system. (A) In vivo RNA interference activity in the cmr4α mutant strains determined by artificial mini-CRISPR-based reporter gene assay. The chromosomal lacS gene was used as the reporter gene. pSe-Rp: reference plasmid; pAC-SS1: an artificial mini-CRISPR plasmid carrying S1 spacer of the lacS gene. (B) Invading plasmid silencing activity of the cmr4α mutant strains. pSeSD1: the reference plasmid; pS10i: an invading plasmid carrying a target sequence of spacer 10 in CRISPR locus 2 in S. islandicus REY15A; Δβ: the wild-type strain; other strains: mutants carrying the specified point mutation of Cmr4α; 4GA: G244AG245AG250AG252A substitutions.

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  Figure 5. crRNA distribution pattern and activities of Cmr-α_4αH16A and Cmr-α_4αD83A. (A) Schematic map of the shorter (carrying a 40-nt crRNA and three Cmr4 subunits) and the longer (carrying a 46-nt crRNA and four Cmr4 subunits) Cmr-α complexes and their RNA cleavage products. Asterisk symbol at each RNA end indicates radio-labeling. (B) crRNA distributions of WT (wild-type Cmr-α), 4αH16A and 4αD83A represent the Cmr-α effectors carrying the indicated Cmr4α mutation (i.e., Cmr-α_4αH16A and Cmr-α_4αD83A). Activities of the three Cmr-α complexes: tgRNA cleavage (C), ssDNA cleavage (D) and cOA synthesis (E). Cleavage assay was conducted for the time periods indicated in the figures; smaller fragments in panel C and D panels represent cleavage products. The sizes of RNA cleavage products in 6-nt periodicity are indicated. None: substrate only, no Cmr complex was added; duplex: duplex of crRNA and substrate; cOA: cyclic oligoadenylates.