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Foreign DNA capture during CRISPR–Cas adaptive immunity

Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40 base pair (bp) lengths into clustered regularly interspaced short palindromic repeats (CRISPR) loci as spacer segments1–6. The universally conserved Cas1–Cas2 integrase complex catalyzes spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7–13. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33 nucleotide (nt) protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′–OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo2–4. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

and splays the ends of the protospacer to allow each terminal nucleophilic 3′-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo 2 -4 . Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.
CRISPR loci are defined by repetitive elements that are separated by similarly-sized spacer sequences acquired from foreign DNA during the adaptation stage of CRISPR-Cas adaptive immunity 6 , 14 . CRISPR transcripts generated from the loci assemble with Cas proteins to detect and cleave foreign nucleic acids bearing sequence complementarity to the spacer segment 1 , 5 , 15 -19 . In E. coli, expression of the Cas1-Cas2 protein complex triggers acquisition of new 33 bp spacers at the A/T-rich leader end of the CRISPR locus 7 -10 , 20 . How the Cas1-Cas2 complex selects 33 bp protospacers of variable sequences and activates the 3′-OH ends for integration remains unknown. As the Cas1-Cas2 complex is sufficient to initiate spacer acquisition and adaptation of the CRISPR-Cas immune system, we hypothesized that the protein complex alone must provide the structural basis for the unknown mechanism of spacer length determination.
To determine how protospacer variation influences the efficiency of Cas1-Cas2-mediated spacer acquisition, we used an in vitro integration assay to test versions of a 33 bp sequence with constant overall length but different 3′ single-stranded overhang lengths 12 . The protospacer sequence is derived from the M13 bacteriophage genome and is highly acquired into the E. coli CRISPR locus after infection 8 . Unexpectedly, protospacers with overhanging 3′ nucleotides are strongly preferred by the Cas1-Cas2 complex over a completely doublestranded 33 bp protospacer ( Fig. 1a and Extended Data Fig. 1a,b). Single-stranded DNA and substrates with 5′ overhangs are poor substrates for integration, highlighting the ability of Cas1-Cas2 to select specific DNA substrates prior to integration 12 . The most preferred protospacer DNA for in vitro integration consists of five overhanging nucleotides on each 3′ end (Extended Data Fig. 1). To determine the molecular basis of Cas1-Cas2 protospacer capture, we assembled Cas1-Cas2 complexes with the preferred protospacer substrate and determined crystal structures of the complex in the presence and absence of Mg 2+ at 3.0 Å and 3.2 Å resolution, respectively (Extended Data Fig. 2 and Extended Data Table 1).
The structures reveal a hexameric protein architecture comprising four copies of Cas1 and two copies of Cas2, in which the protospacer spans the central Cas2 dimer and terminates within individual Cas1 subunits on each end of the complex (Fig. 1b). Structural superposition of the Cas1-Cas2 complex with and without bound DNA reveals a DNAinduced change in Cas1 subunit orientation in which each Cas1 dimer rotates ~10° in opposing directions against the central Cas2 hub (Extended Data Fig. 3a,b). Cas1-Cas2 protospacer capture positions each single-stranded protospacer 3′ end within a channel leading directly to a Cas1 active site. Simulated annealing omit maps show clear electron density for the double-helical region and the five-nucleotide overhangs on each end of the protospacer (Extended Data Fig. 4a-c). The constrained protein channel guiding each DNA strand from its double-helical region to the single-strand-accommodating Cas1 active site explains the specificity of Cas1-Cas2 for five-nucleotide 3′ overhang substrates ( Fig. 1a and Extended Data Fig. 1). Two of the four Cas1 subunits, colored green in Fig. 1b, are not occupied with the protospacer 3′ ends and are likely non-catalytic since the 3′-OH nucleophile and the scissile phosphodiester bond of the target DNA must be in the same active site for direct nucleophilic integration.
In the active sites, the 3′ terminal base is involved in a stacking interaction with Y217 that positions the nucleophilic 3′-OH ends of the protospacer near the conserved metal-binding residues E141, H208 and D221 (Fig. 1c). Although we cannot assign density for Mg 2+ in the active sites, these three residues have been shown previously to coordinate a Mn 2+ ion in the active site of Cas1 from P. aeruginosa 21 . Furthermore, alanine mutations at these positions disrupt in vivo spacer acquisition 7 , 8 , 10 . Thus, the observed positioning of the 3′-OH nucleophiles and catalytic residues likely represents the active configuration of the nucleoprotein complex immediately prior to spacer integration.
All interactions between Cas1-Cas2 and protospacer DNA involve coordination of the phosphate-backbone rather than base-specific contacts, consistent with the variable sequence selection of protospacers that is essential for resistance to diverse foreign sequences 2 -4 . Two central regions of the Cas1-Cas2 complex, which we term the Arginine Clamp and the Arginine Channel, stabilize the protospacer ( Fig. 2a-d). The Arginine Clamp interacts with the middle of the duplex region where four Arg residues coordinate each DNA strand: Cas1 R41 and Cas2 R16, R77, R78 (Fig. 2c). Reverse charge mutations of Cas1 R41 and Cas2 R16 and R78 drastically reduce spacer acquisition in vivo, whereas the Cas2 R77E mutant functions similar to wild-type (WT) Cas2 (Fig. 2e). Thus, Cas1 R41, Cas2 R16 and R78 are the key constituents of the Arg clamp. The contribution of Cas2 to protospacer DNA binding supports the previous hypothesis that the main function of Cas2 is to form a non-catalytic scaffold within the Cas1-Cas2 complex 10 .
Cas1 residues R66, R84, R245 and R248 line the Arginine Channel that stabilizes the junction where the duplex region terminates and the ssDNA overhang enters the active site. Reverse charge mutation of each arginine lining the Arginine Channel disrupts spacer acquisition in vivo (Fig. 2e). In addition, purified Cas1 R59D or R66D proteins complexed with WT Cas2 are highly defective in integrating 33 bp duplex or five-nucleotide overhang protospacer substrates in vitro (Fig. 2f). Fluorescence polarization assays demonstrate that the mutant complexes exhibit dramatically reduced affinity for protospacer DNA, highlighting the critical role of this part of the Cas1-Cas2 complex for protospacer capture and complex stability (Fig. 2g).
The Cas1-Cas2-DNA crystal structures uncover a protein wedge that terminates the protospacer dsDNA region and allows single-stranded DNA overhangs to enter the Arginine Channel. A stacking interaction of the 5′ terminal base (adenine 6 in Fig. 3a,b) with Y22 of Cas1 stabilizes protospacer duplex unwinding, directing each single-stranded 3′ overhang to sharply bend ~90° away from the duplex and into the active site channel (Fig. 3b). A mutation of Y22 to alanine reduces spacer acquisition in vivo whereas a phenylalanine mutation has near WT levels of acquisition, consistent with a specific role for Cas1 Y22 base-stacking in protospacer strand splaying (Fig. 3c). Sequence alignment of representative Cas1 proteins in Type I CRISPR systems reveals that Y22 is not universally conserved in other bacteria, suggesting that additional or different Cas1 residues may stabilize the splayed ends in other CRISPR-Cas systems (Extended Data Fig. 5).
The observed stacking interaction raises the possibility that fully duplexed protospacers are separated by Cas1 Y22, thereby displacing the 5′ end of the duplex, we term the nonnucleophilic strand, from the nucleophilic strand carrying the 3′-OH. DNA transposases and retroviral integrases also utilize end fraying to isolate the reactive DNA strands for chemistry within enzyme active sites 22 -24 . To test this potential activity of Cas1-Cas2, we introduced an increasing number of mismatches at the ends of the 33 bp protospacer to disrupt end base pairing and assayed their potential for in vitro integration ( Fig. 3d and Extended Data Fig.   6a,b). Similar to the 3′ overhang substrates, the 4 and 5 nt frayed ends are highly preferred, presumably due to the lower energy required for capture of these substrates compared to perfectly duplexed ends (Fig. 3d). The complex containing the Cas1 Y22A mutant regains marginal activity with substrates containing 5 or 6 nt splayed ends, suggesting that Y22 steers the non-nucleophilic DNA strand away from the active site (Fig. 3d). Notably, the displaced non-nucleophilic strand is not cleaved into a shorter fragment by Cas1-Cas2, as the protospacer ends are not processed during integration (Extended Data Fig. 6c).
To determine the trajectory of the displaced non-nucleophilic strand after end-splaying, we crystallized Cas1-Cas2 with a protospacer with five-nucleotide frayed ends on both sides (Fig 3a,b). The electron density at the fork is similar to the structures described above, except we observe the first nucleotide of the displaced non-nucleophilic strand pointing in the opposite direction from the nucleophilic ssDNA strand. Clear electron density is not observed for the remaining nucleotides of the displaced strand, indicating that they are not stabilized by the complex.
An alternative crystal form grown in the presence of Mg 2+ reveals secondary Cas1-DNA interactions that provide additional insight into the mechanism of Cas1-Cas2 genomic DNA target binding and subsequent integration. In addition to the two Cas1 "catalytic" active sites carrying the 3′-OH ends of the protospacer, the "non-catalytic" Cas1 active sites interact with the protospacer DNA from a symmetry mate, revealing a possible coordination of the target DNA during integration ( Fig. 4a and Extended Data Fig. 7a). The non-catalytic Cas1 engages the DNA minor groove by contacts with α helix 7, causing a slight kink on the DNA compared to our alternative crystal form lacking Mg 2+ (Extended Data Fig. 7b). A close-up of the active site shows continuous density for Mg 2+ with E141, H208, D221 and a phosphate backbone of the presumed target DNA, capturing a snapshot of scissile phosphodiester bond coordination prior to integration (Fig. 4a).
Because integration must occur in the active site that coordinates the 3′-OH of the protospacer DNA, we modeled the protein-DNA interactions from the non-catalytic Cas1 active sites into the catalytic Cas1 active sites. This reveals the positioning of the nucleophilic 3′-OH of the protospacer ends for attacking the scissile phophodiester bond in the modeled DNA (Fig. 4b, c). Further work will be needed to shed light on how the complex specifically recognizes the leader-repeat region of the CRISPR locus for integration, as recently observed in vitro 11 -13 .
Together, these data explain key aspects of Cas1-Cas2 integrase-mediated acquisition of new DNA into bacterial genomes. First, we show that the substrates for integration are double-stranded DNA. Importantly, however, optimal substrates include a central 23 base pair helical region flanked by five single-stranded nucleotides on each 3′ end. If substrates for CRISPR integration come from ssDNA products of RecBCD, as recently suggested, they must somehow anneal or otherwise become double stranded prior to Cas1-Cas2 capture 20 . It remains unclear how the Cas1-Cas2 complex recognizes the AAG protospacer adjacent motif (PAM) during protospacer selection, since the terminal nucleotides containing the 3′-OH nucleophiles are coordinated similarly in the Cas1 active sites (Fig. 1). Second, the Cas1-Cas2 integrase architecture specifies the precise length of integrated DNA, ensuring uniformity of spacer lengths within CRISPR loci. Finally, the structure-based model of DNA target sequence positioning suggests that in addition to catalyzing the integration reaction, Cas1 plays a role in binding the target CRISPR locus. Target binding could possibly disrupt the structural symmetry observed in the crystal structure to coordinate the sequence-specific integration reactions at the leader-end of the CRISPR locus. Insights into target site recognition may offer strategies for altering or enhancing integration site specificity, with implications for use of the Cas1-Cas2 integrase as a genome-modifying technology.

Cas1, Cas2 and DNA preparation
The Cas1 and Cas2 proteins from E. coli K12 (MG1655) were cloned and separately purified as previously described 10 . Single-stranded DNA oligonucleotides purchased from Integrated DNA Technologies were annealed in 20 mM HEPES-NaOH, pH 7.5, 25 mM KCl, 10 mM MgCl 2 by heating at 95 °C for 3 min and slow cooling to room temperature. The pCRISPR DNA target for in vitro integration was constructed as previously described 12 .

In vivo acquisition and in vitro integration assays
The in vivo acquisition assays were performed as previously described 7 . The in vitro integration reactions were conducted as previously described with slight modifications 12 .
After pre-incubation of equimolar Cas1 and Cas2 at 4 °C, 100 nM of the resulting Cas1-Cas2 complex was incubated with 100 nM protospacer DNA for an additional 10-15 min at room temperature. The integration reaction was activated by the addition of 300 ng (~5 nM) pCRISPR, incubated at 37 °C for 1 h and quenched with DNA loading buffer supplemented with EDTA at a final concentration of 20 mM. The reaction products were analyzed on 1.5% agarose gels. Percent integration activity values were determined by quantifying the band intensity of the relaxed pCRISPR product and dividing over the intensity of all bands detected by Image Lab Software (Bio-Rad). We note that the integration activity could be a mixture of half-site and full-site integration products, as described previously 12 . X-ray diffraction data were collected under cryogenic conditions at beamline 8.3.1 at the Lawrence Berkeley National Laboratory Advanced Light Source. Initial phases were obtained by sequential molecular replacement using individual protein components of the Cas1-Cas2 apo structure (PDB 4P6I) as search models. Following initial placement of two Cas1 dimers and a dimer of Cas2, phases were improved by performing one round of rigid body refinement in PHENIX 25 . The resulting maps showed clear unbiased density for protospacer DNA, and subsequent model building was performed through iterative rounds of building in Coot 26 and refinement in PHENIX with NCS restraints on the protein subunits.

Complex formation, crystallization and structure determination
The asymmetric unit of the three structures contains one copy of the Cas1-Cas2 complex bound to protospacer DNA. Statistics for the final crystal structures are reported in Extended Data Table 1. The final structures are missing clear density for the loop connecting α6 and α7 of Cas1. We assume this loop to be highly disordered as it is also not observed in the apo E. coli Cas1 crystal structure (PDB 3NKD) and the apo Cas1-Cas2 complex (PDB 4P6I) 10 , 27 .

Fluorescence polarization
Fluorescence polarization assays were performed in 20 mM HEPES-NaOH, pH 7.5, 25 mM KCl, 5 mM EDTA, 1 µg ml −1 BSA and 1 mM DTT. Cas1-Cas2 were complexed and purified over gel filtration for all binding assays. The 3′-fluorescein labeled DNA substrate was added to the protein solution at a final concentration of 5 nM and the DNA-protein mixture was allowed to incubate for 30 min at 22 °C. Measurements were made by excitation at 485 nm and monitoring emission at 535 nm. Data were fit to a binding isotherm to obtain K d . Each experiment was conducted in triplicates and error bars represent the standard deviation.

Sequence alignment
The cas1 sequences were obtained from the National Center for Biotechnology Information (NCBI) Gene Data Bank. A representative cas1 from each CRISPR Type I subtype were Extended Data Figure 6. Integration of protospacer substrates with splayed ends a, Representative agarose gel of in vitro integration reactions using increasing lengths of splayed ends. The average percent integration of three independent experiments is plotted in Fig. 3d. b, Sequences of protospacers used in the integration assays in a. c, A 12% denaturing polyacrylamide gel of protospacers after incubation with Cas1-Cas2 for 1 h at 37 °C in integration assay buffer conditions. The indicated DNA substrates are radiolabeled at the 5′ end. Supplementary Information contains the full images for a and c.
Extended Data Figure 7. Crystallographic packing of the complex bound to Mg 2+ a, View of the symmetry mates (gray) contacting the non-catalytic Cas1 subunits (green). b, Superposition of our two crystal structures, with or without Mg 2+ , show a slight DNA kink in the structure bound to Mg 2+ (dotted box). This region contacts α helix 7 of a symmetry mate, as described in the text.
Extended Data Table 1 Summary of X-ray crystallography data collection and refinement.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.