In most animals, the adaptive immune system creates specialized cells that adapt to efficiently fight off any viruses or other pathogens that have invaded. Bacteria (and another group of single-celled organisms called archaea) also have an adaptive immune system, known as CRISPR-Cas, that combats viral invaders. This system is based on sections of the microbes' DNA called CRISPRs, which contain repetitive DNA sequences that are separated by short segments of ‘spacer’ DNA. When a virus invades the cell, some viral DNA is incorporated into the CRISPR as a spacer. This process is known as adaptation. CRISPR-associated proteins (or ‘Cas’ proteins) then use this spacer to recognize and mount an attack on any matching invader DNA that is later encountered.

Exactly how a spacer is inserted into the correct position in the CRISPR array during adaptation remains poorly understood. However, it is known that two CRISPR proteins called Cas1 and Cas2 play essential roles in this process.

Rollie et al. took Cas1 proteins from a bacterial cell (Escherichia coli) and an archaeal species (Sulfolobus solfataricus) and added them to branched DNA structures in the laboratory. These experiments revealed that Cas1 from both organisms can break the DNA down into smaller pieces. Cas2, on the other hand, is not required for this process. This ‘disintegration’ reaction is the reverse process of the ‘integration’ step of adaptation where the CRISPR proteins insert the invader DNA into the CRISPR array.

Rollie et al. also found that the disintegration reaction performed by Cas1 takes place on specific DNA sequences, which are also the sites where Cas1 inserts the spacer DNA during adaptation. Therefore, by examining the disintegration reaction, many of the details of the integration step can be deduced.

Overall, Rollie et al. show that selection by Cas1 plays an important role in restricting the adaptation process to particular DNA sites. The next step will be to use the disintegration reaction to examine the DNA binding and manipulation steps performed by Cas1 as part of its role in the adaptation of the CRISPR system.

The CRISPR-Cas system is an adaptive immune system present in many archaeal and bacterial species. It provides immunity against viruses and other mobile genetic elements mediated through sequence homology-directed detection and destruction of foreign nucleic acid species (reviewed in Sorek et al., 2013; Barrangou and Marraffini, 2014; van der Oost et al., 2014). Organisms with an active CRISPR-Cas system encode one or more CRISPR loci in their genomes. These comprise a leader sequence followed by an array of short, direct, often palindromic repeats interspersed by short ‘spacer’ sequences, together with a variable number of CRISPR associated (cas) genes. Spacers are derived from mobile genetic elements and constitute a ‘memory’ of past infections. The CRISPR locus is transcribed from the leader, generating pre-CRISPR RNA (pre-crRNA) that is subsequently processed into unit length crRNA species and loaded into large ribonucleoprotein complexes. These complexes, known as ‘Interference’, ‘Effector’ or ‘Surveillance’ complexes, utilize crRNA to detect and degrade cognate invading genetic entities, providing immunity against previously encountered viruses and plasmids.

The process of foreign DNA capture and integration into the CRISPR locus is known as ‘Acquisition’ or ‘Adaptation’ (reviewed in Fineran and Charpentier, 2012; Heler et al., 2014). This process underpins the whole CRISPR-Cas system, but is the least well understood aspect. Fundamentally, acquisition can be separated into two steps: the capture of new DNA sequences from mobile genetic elements, followed by the integration of that DNA into the host genome. New spacers are incorporated proximal to the leader sequence and result in the duplication of the first repeat (Goren et al., 2012; Yosef et al., 2012). The leader sequence proximal to the repeat is important for integration, but transcription of the locus is not required (Yosef et al., 2012). New spacers are frequently incorporated in both possible orientations (Shmakov et al., 2014). The integration process in Escherichia coli results in staggered cleavage of the first CRISPR repeat, where the 3′-end of one strand of the incoming DNA is joined to the end of the CRISPR repeat in a trans-esterification (TES) reaction, with another TES reaction occurring on the other strand (Diez-Villasenor et al., 2013) (Figure 1, numbered yellow arrows). Intermediates in this reaction have been observed in E. coli, and the sequence of the leader-repeat1 junction is important for the activity (Arslan et al., 2014). The order of the two integration steps shown in Figure 1 is not yet known, although it has been suggested that the reaction on the minus strand (site 2) occurs first in E. coli (Nuñez et al., 2015). The sequence at site 2 is flanked by the end of the first repeat and the first spacer, and is therefore inherently less well conserved. In E. coli, the last nucleotide of the newly copied repeat is derived from the first nucleotide of the incoming spacer, which imposes further sequence requirements on that system (Swarts et al., 2012).

Figure 1

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Although shown in Figure 1 as fully double-stranded, the incoming spacer DNA could have a partially single-stranded character. Recent work by Sorek and colleagues has shown that the E. coli RecBCD helicase–nuclease complex, which processes DNA double-strand breaks for recombination and degrades foreign DNA, is implicated in the generation of viral DNA fragments captured by Cas1 and incorporated into the CRISPR locus as new spacers (Levy et al., 2015). This confirms previous observations linking Cas1 with RecBCD (Babu et al., 2011) and raises some intriguing questions as RecBCD generates ssDNA fragments asymmetrically, generating fragments tens to hundreds of nucleotides long from the 3′ terminated strand and much longer fragments from the 5′ terminated strand (reviewed in Dillingham and Kowalczykowski, 2008). The Cas4 enzyme, which is a 5′ to 3′ ssDNA exonuclease (Zhang et al., 2012; Lemak et al., 2013), may provide ssDNA fragments for Cas1 in systems lacking RecBCD. However, it is difficult to see how two integration reactions could occur without two 3′ hydroxyl termini (Figure 1) and half-site integration is not observed with a ssDNA substrate (Nuñez et al., 2015). Potentially, the ssDNA fragments generated by these nucleases may re-anneal and experience further processing to generate partially duplex molecules of defined size prior to integration by Cas1.

Adaptation requires the products of the cas1 and cas2 genes in vivo and these are the most universally conserved of all the cas genes (Makarova et al., 2006). Cas1 is a homodimeric enzyme with a two-domain structure and a canonical metal dependent nuclease active site in the large domain formed by a cluster of highly conserved residues (Wiedenheft et al., 2009) (Figure 1C). E. coli Cas1 has nuclease activity in vitro, with activity observed against double- and single-stranded DNA and RNA substrates (Babu et al., 2011). Some specificity was observed for branched DNA substrates, in particular for DNA constructs resembling replication forks (Babu et al., 2011). Initial biochemical analyses of a panel of archaeal Cas2 enzymes revealed an endonucleolytic activity against ssRNA substrates that could be abrogated by mutation of conserved residues (Beloglazova et al., 2008). In contrast, Cas2 from Bacillus halodurans has been shown to be specific for cleavage of dsDNA substrates (Nam et al., 2012). Recently however, Doudna and colleagues demonstrated that E. coli Cas1 and Cas2 form a stable complex in vitro and that the ‘active site’ of Cas2 was not required for spacer acquisition, suggesting that Cas2 may not have a catalytic role in spacer acquisition in vivo (Nuñez et al., 2014). It is probable that Cas2 acts as an adaptor protein, either bringing two Cas1 dimers together or mediating interactions with other components necessary for spacer acquisition. Recently, Nunez et al. demonstrated that E. coli Cas1 can integrate a protospacer into a supercoiled plasmid in vitro, in a reaction stimulated by Cas2. Integration events were observed at the boundaries of most CRISPR repeats and in other areas of the DNA close to palindromic regions, implicating a role for palindromic DNA structure in the adaptation process (Nuñez et al., 2015).

In order to further mechanistic understanding of the spacer acquisition process, we have undertaken a systematic analysis of the underlying biochemistry. We demonstrate that Cas1 catalyses TES of branched DNA substrates efficiently in vitro in a reaction that represents the reverse- or disintegration of an incoming spacer from the CRISPR locus. The disintegration reactions catalysed by diverse integrases have proven a powerful model system for the understanding of the mechanism of integration. For Cas1, the reaction is strongly sequence dependent with the specificity matching the predicted integration site 1 for both E. coli and Sulfolobus solfataricus Cas1, and does not require Cas2.

Studies of the CRISPR spacer acquisition process in vivo have yielded many key insights, but they are complicated by the fact that it is very difficult to separate the two distinct steps of spacer capture and spacer integration. Consequently we still do not have a clear understanding of the roles of Cas1, Cas2 and host proteins in the acquisition mechanism. In this study we investigated the integration reaction by focusing on the biochemistry of the Cas1 protein from S. solfataricus and E. coli. Efficient TES of branched DNA substrates with a 5′-flap or duplex arm is clearly possible for both S. solfataricus and E. coli Cas1 in vitro. This is a very precise reaction requiring attack by a 3′-hydroxyl at the branch point, generating a perfect DNA duplex. The reaction almost certainly represents the disintegration reaction that is the reverse of the spacer integration step, as observed for many integrases and transposases where it represents a very useful means to study the underlying integration mechanism (Gerton et al., 1999). Evidence for a disintegration activity was recently described by Doudna and colleagues for EcoCas1, but the activity observed was relatively weak, most likely because the branched substrate studied had a non-optimal DNA sequence around the branch point (Nuñez et al., 2015).

For the CRISPR adaptation process in vivo, integration occurs at the junction between the first repeat and the leader sequence, which immediately suggests a role for sequence specificity in the reaction. It has also been suggested that CRISPR repeat sequences, which are often palindromic, form four-way DNA junctions in supercoiled DNA, acting as a recognition signal for Cas1, a possibility that is supported by the observation that EcoCas1 can cut four-way DNA junctions in vitro (Babu et al., 2011), and the finding that spacer integration in a plasmid lacking a CRISPR locus occurs preferentially next to a palindromic site (Nuñez et al., 2015). However, a palindrome alone is not sufficient to support spacer insertion in E. coli in vivo (Arslan et al., 2014), and this also holds for the type II CRISPR system of Streptococcus thermophilus (Wei et al., 2015), suggesting that local sequence helps determine the integration site. Furthermore, some CRISPR repeat families, including many in archaea, have little or no palindromic nature and thus cannot form stable hairpin structures (Kunin et al., 2007). Cas1 therefore might be expected to act as a sequence specific integrase, although local DNA structure could also play a part.

In support of this hypothesis, both S. solfataricus and E. coli Cas1 catalyse a disintegration reaction with distinct, sequence specific properties. In particular, there is a clear preference for a guanine at position +1, corresponding to the first nucleotide of the repeat, suggesting that this residue is recognised specifically in the active site of Cas1. The specificity is particularly strong for EcoCas1, consistent with the presence of a guanine in the repeat sequence at the +1 site in both plus and minus strands. Preference for a guanine at the +1 nucleotide for EcoCas1 catalysed integration events has also been observed (Nuñez et al., 2015). For SsoCas1, a guanine at this position was preferred over a cytosine, which is the nucleotide present at the +1′ position on the minus strand, by a factor of five. Although the −1 position might also be expected to play a role in the selection of integration sites, deep sequencing data for integration catalysed by EcoCas1 revealed no sequence preference at this position (Nuñez et al., 2015). In agreement with this finding, we observed little evidence for sequence discrimination at the −1 position for the disintegration reactions catalysed by either enzyme, with the exception that EcoCas1 disfavours cytosine at this position (Figure 6B). A cytosine at position −1 is the expected residue on the minus strand, suggesting that the disintegration reaction may better reflect the reversal of integration at site 1 in the leader-repeat junction. Deep sequencing data for integration reactions catalysed by EcoCas1 in vitro did reveal a marked preference for a guanine at position −2 in the integration site (Nuñez et al., 2015). This corresponds well with the −2 position in the plus strand, which is part of the leader sequence and is a guanine in E. coli, but cannot hold for the minus strand where the −2′ position is inherently variable in nature. Disintegration of substrates mimicking the integration intermediates relevant for the integration of spacer 3 into the E. coli CRISPR array reinforce these conclusions, with site 1 on the plus strand processed significantly more quickly than site 2 on the bottom strand (Figure 9). Taken together, both the disintegration specificity and the deep sequencing data for integration support the hypothesis that integration is targeted to the leader-repeat 1 junction on the plus strand at least in part by the inherent sequence specificity of Cas1, which presumably involves specific recognition of these bases within the active site of the enzyme (Figure 10).

Figure 10

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For E. coli integration reactions in vitro, a marked preference for cytosine over thymine at the 3′ end of the protospacer was observed (Nuñez et al., 2015). Furthermore, protospacers with a cytosine at the 3′ end were preferentially incorporated into the minus strand at the junction between repeat 1 and spacer 1. These data are consistent with the requirement for protospacers to supply the final cytosine of the repeat on the minus strand during integration (Swarts et al., 2012). The deep sequencing data also revealed a marked preference for adenine over thymine at the 3′ end of the protospacer (Nuñez et al., 2015). For disintegration by EcoCas1, we observed a clear preference for adenine or cytosine at the equivalent position, whilst thymine did not support the disintegration reaction (Figure 8). Thus EcoCas1 appears to recognise the nucleotide at the 3′ end of the incoming DNA, although no such discrimination was observed for SsoCas1. A dinucleotide sequence ‘AA’ motif over-represented at the 3′ end of protospacers incorporated in E. coli strain BL21 has been described previously (Yosef et al., 2013).

Although the CRISPR spacer integration system has been compared to the integration and transposition reactions carried out by mobile genetic elements, there is one key difference in the two processes—the length of DNA integrated. The persistence length of DNA, the distance over which it behaves as a fairly rigid rod, is estimated as 35–50 nm (100–150 bp) under conditions found in cells (Hagerman, 1988; Brinkers et al., 2009). This means that the two ends of a viral genome of several kilobases can be looped around and brought close together relatively easily, but a new spacer of 30–40 base pairs of dsDNA cannot be manipulated in the same way. Considering the scheme in Figure 1, the molecular origami required to achieve the second TES reaction looks challenging. Several related enzymes, including Mu transposase (Savilahti et al., 1995), V(D)J recombinase (Ramsden et al., 1996) and HIV integrase (Gerton et al., 1999) are known to disrupt base pairing of DNA substrates and make sequence-specific contacts during the integration reaction. It is likely that Cas1 also manipulates the local DNA duplex structure, which may help in positioning the DNA strands correctly for the TES reaction. The observation that the incoming DNA (the 5′-flap in the disintegration reaction) can be single stranded, partially or fully duplex in nature suggests that there is some flexibility in the recognition of the incoming spacer. This is also consistent with the recent observation of a link between RecBCD, which generates ssDNA products, and Cas1 in E. coli (Levy et al., 2015). There is no formal requirement that the protospacer should be fully duplex in nature, although current understanding of the integration reaction requires that new spacers have two intact 3′-ends for the two integration reactions so must be at least partially duplex in nature. Many integrases process the ends of the integrated DNA using a nuclease activity, which occurs at the same active site as the integrase activity (Gerton et al., 1999). There is no reason to expect that Cas1 will differ in that regard, and indeed the reaction products of the RecBCD nuclease are on average much longer than the DNA molecules integrated by Cas1, suggesting the requirement for further processing.

In conclusion, we have shown that Cas1 from both S. solfataricus and E. coli have robust TES activities in vitro which reflect the reversal, or disintegration, of the integration reaction. Disintegration is strongly sequence specific, and the specificity fits with the expected sequence for the plus strand at the leader-repeat junction (Figure 9). This site is the logical place for the initiation of integration, as it has a unique, defined sequence, in contrast to the repetitive and more variable sequence found at the second integration site on the minus strand. Doudna and colleagues recently proposed a model based on an initial attack at site 2 on the minus strand (Nuñez et al., 2015). However, this preference was only significant for spacers with a cytosine at the 3′ terminus, and does not explain the marked preference observed by the authors for a guanine at position +2, which is a feature of the positive strand. Future studies of both the forward and reverse integration reactions catalyzed by Cas1 will help to address these issues and delineate the mechanism of spacer integration in the CRISPR system.

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Author details

1. Clare Rollie

   Biomedical Sciences Research Complex, University of St Andrews, St Andrews, United Kingdom

   Contribution

   CR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Stefanie Schneider

   Faculty of Medicine, Institute of Cell Biology, University of Duisburg-Essen, Essen, Germany

   Contribution

   SS, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

3. Anna Sophie Brinkmann

   School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom

   Contribution

   ASB, Acquisition of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Edward L Bolt

   School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom

   Contribution

   ELB, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Malcolm F White

   Biomedical Sciences Research Complex, University of St Andrews, St Andrews, United Kingdom

   Contribution

   MFW, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   mfw2@st-and.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-1543-9342

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