Target DNA recognition and cleavage by a reconstituted Type I-G CRISPR-Cas immune effector complex

Abstract

CRISPR-Cas immune systems defend prokaryotes against viruses and plasmids. CRISPR RNAs (crRNAs) associate with various CRISPR-associated (Cas) protein modules to form structurally and functionally diverse (Type I-VI) crRNP immune effector complexes. Previously, we identified three, co-existing effector complexes in Pyrococcus furiosus -Type I-A (Csa), Type I-G (Cst), and Type III-B (Cmr)-and demonstrated that each complex functions in vivo to eliminate invader DNA. Here, we reconstitute functional Cst crRNP complexes in vitro from recombinant Cas proteins and synthetic crRNAs and investigate mechanisms of crRNP assembly and invader DNA recognition and destruction. All four known Cst-affiliated Cas proteins (Cas5t, Cst1, Cst2, and Cas3) are required for activity, but each subunit plays a distinct role. Cas5t and Cst2 comprise a minimal set of proteins that selectively interact with crRNA. Further addition of Cst1, enables the four subunit crRNP (Cas5t, Cst1, Cst2, crRNA) to specifically bind complementary, double-stranded DNA targets and to recruit the Cas3 effector nuclease, which catalyzes cleavages at specific sites within the displaced, non-target DNA strand. Our results indicate that Type I-G crRNPs selectively bind target DNA in a crRNA and, protospacer adjacent motif dependent manner to recruit a dedicated Cas3 nuclease for invader DNA destruction.

Keywords: CRISPR; Cas; Cas3; Cst; Pyrococcus furiosus; Type I-G.

Fig. 1

Pyrococcus furiosus cas gene organization and annotation. Genes encoding the crRNA biogenesis protein [(cas6), grey], predicted CRISPR DNA uptake proteins [(cas 1, cas2, and cas4), grey], and three effector modules—Type III-B [(Cmr), blue], Type I-G [(Cst), yellow (boxed), and Type I-A (Csa), green] are shown. The annotated superfamily designations (e.g., cas5, cas7, etc.) are denoted below each gene name

Fig. 2

Cas protein requirements for crRNA binding. a SDS-PAGE showing purified recombinant Cas5t, Cst2, Cst1, and Cas3 and molecular mass marker proteins (M). b Native gel electrophoresis of 3′-radiolabeled crRNA incubated in the absence (−) or presence (+) of Cas5t, Cst2, Cst1, and/or Cas3 proteins as indicated. The positions of unbound crRNA (crRNA), non-specific binding associated with Cas3 (asterisk) and Cst complexes (crRNP, bracket) are highlighted

Fig. 3

Key crRNA features for assembly of Cst crRNP complexes. a Gel mobility shift assay showing Cst crRNP formation with 2, 3, or 4 Cst Cas proteins as indicated and 3′-radiolabeled (³²P) crRNA containing either a wild-type 5′ tag (black) or mutated 5′ tag (red). b Gel mobility shift assay showing Cst crRNP formation with 2,3, or 4 Cst Cas proteins and crRNA bearing either 5′ hydroxyl and 3′ phosphate [(5′ OH/3′ P), wild type shown in schematic], 5′ phosphate and 3′ hydroxyl (5′ P/3′ OH), or 5′ hydroxyl and 3′ hydroxyl (5′ OH/3′ OH). The guide sequence (orange) is wild type in all cases. Unbound crRNAs (crRNA) and Cst complexes (crRNP, bracket) are indicated on right

Fig. 4

Identification of direct interactions between crRNA and Cst proteins via UV-crosslinking. a Gel mobility shift assay showing crRNPs formed under crosslinking conditions in the presence (+) or absence (−) of individual Cst proteins, Cas5t + Cst2, or Cas5t + Cst2 + Cst1, as indicated. b SDS polyacrylamide gel (G250) of Cst Cas proteins, RNAse T1, and/or RNase A in each reaction plus corresponding autoradiographic images (Rad) identifying crRNA-interacting Cst Cas proteins (arrowheads). Schematic above each planel indicates the ³²P- and 4-thiouridine-labeled RNA used in each experiment [5′ tag sequence (black), guide sequence (orange), radiolabeled region (asterisks)]

Fig. 5

Components of Cst crRNP essential for DNA targeting. a Schematics of double-stranded DNA substrates used in the assay. Wild-type protospacer (red), wild-type PAM (blue), target strand (complementary to crRNA) and non-target strand of DNA, and 5′ tag (black) and guide (red) of crRNA (assembled in Cst crRNP) are labeled. b Gel mobility shift assay with either no protein (−) or Cst crRNP formed with either Cas5t + Cst2, Cas5t + Cst2 + Cst1 (black triangle), Cas5t + Cst2 + Cst1 + Cas3 (grey triangle), all 4 Cst proteins without crRNA, and Cas3 + crRNA in the absence of Cst proteins. Asterisk indicates non-specific binding of DNA by Cst1. c Corresponding DNA cleavages (asterisk, bracket) of samples in b. 5′-radiolabeled DNA size-standard (Affymetrix, low molecular weight marker, 10–100 bases) is labeled (M). The top line of b, c indicates the location of the ³²P-label in either the non-target or target strand

Fig. 6

PAM sequence requirements for DNA recognition and destruction. a Schematics of double-stranded DNA substrates used in the assay: crRNA (top) containing 5′ tag (black) and guide (red), target and non-target strands of double-stranded DNA with wild-type protospacer (red) plus either wild-type PAM (blue), C-G PAM (double-stranded but mutant in both strands), C-C PAM (single-stranded wild-type PAM in non-target strand), or G-G PAM (single-stranded wild-type PAM in target strand). b DNA binding and c DNA cleavages (asterisk) by either no protein (−) or Cst crRNP formed with Cas5t + Cst2 + Cst1 + Cas3 and the specified substrates, as labeled. Non-target strand of DNA is 5′-radiolabeled (³²P) in all cases. Mobility of ³²P-labeled DNA size-standards (in basepairs) are indicated (M)

Fig. 7

DNA cleavages by Cas3 (HD) nuclease map to the protospacer of the non-target strand. a DNA cleavage by no protein (−) or Cst crRNP (Cas5t + Cst2 + Cst1) assembled with either wild-type Cas3 (wt) or mutated Cas3 (mut, HD-AA) from either Pfu or Tko. Non-target strand of DNA is 5′-radiolabeled (³²P). Mobility of ³²P-labeled DNA size-standards (in basepairs) are indicated (M). b Comparison of Pfu Cas3- and Tko Cas3-catalyzed major cleavages (long black arrows) and minor cleavages (short grey arrows) mapped to DNA sequence [PAM (blue), protospacer (red)]. Numbers accompanying arrows indicate size of products. Prominent products present in both digests are marked by an asterisk on the gel a and in the sequence b

Fig. 8

Mechanistic model of Cst crRNP complex assembly, DNA recognition, and cleavage in Pfu. Cas5t (orange) and Cst2 (yellow) interact with 5′ tag (black) and Cst2 interacts with guide (red) of crRNA to form minimal crRNP. Guided by crRNA, Cas5t + Cst2 + Cst1 forms a surveillance complex. Cst1 (purple) scans and interacts with the PAM (blue), enabling the guide sequence of crRNA to base-pair with the target strand of the cognate protospacer (red), displacing the non-target strand and forming an R-loop. The Cas3 nuclease (teal) is recruited to the Cst crRNP/R-loop complex, and the Cas3 nuclease cuts the non-target strand of the protospacer at multiple sites within the R-loop (indicated by arrows)