Memory Upgrade: insights into primed adaptation by CRISPR-Cas immune systems

Abstract

Recently, Künne et al. (2016) demonstrated that degradation products of Cas3 obtained during CRISPR interference fuel priming. In this issue of Molecular Cell, Xue et al. (2016) highlight the conformational changes in Cascade that underpin the priming process when interference is blocked.

Prokaryotes defend against viral infection through an adaptive immune system that relies on CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (cas) genes. CRISPRs are host arrays containing spacer sequences that are derived from foreign DNA. CRISPR-Cas systems are extremely diverse and can be classified into at least five types (types I-VI) (Makarova et al., 2015), type I being the most common. The CRISPR immune response is typically divided into three stages: acquisition, expression and interference. During acquisition new spacers are incorporated into a CRISPR array by the Cas1-Cas2 integrase (Nuñez et al., 2014). During expression, CRISPR arrays are transcribed, processed into CRISPR RNAs (crRNAs) and, in Type I systems, packaged into a surveillance complex called Cascade. During interference, foreign DNA is identified and degraded by the combined action of Cascade and a trans-activating helicase-nuclease, called Cas3. Cascade identifies DNA as foreign if it contains both a sequence complementary to the crRNA (a protospacer) and a protospacer adjacent motif (PAM), avoiding self-targeting due to the lack of a PAM sequence in host CRISPR arrays. The most common PAM in Escherichia coli is the sequence, CTT. Once bound to foreign DNA, Cascade recruits nuclease-active Cas3, which unwinds and degrades the foreign DNA (Redding et al., 2015). The strict requirements for Cascade binding, a PAM and a complementary protospacer, are a potential weakness as mutations in either element provide a simple mechanism for phage to escape CRISPR-Cas immunity. However, the host can restore immunity through a positive feedback loop, called primed adaptation or priming, which rapidly updates the CRISPR arrays with new spacers (Datsenko et al., 2012). To ensure an optimal response, priming is also active with targets containing a correct PAM and fully matching protospacer (Xue et al., 2015). However, this background priming is severely limited by rapid target degradation during interference, which reduces the amount of time in which spacer acquisition can occur (Semenova et al., 2016) but by attenuating or blocking interference escape mutants stimulate priming. Recent studies by Künne et al. (2016) and in this issue by Xue et al. (2016) have provided new insight into primed adaptation. Künne et al. (2016) have revealed that for escape mutants that attenuate interference, degradation products of Cas3 are the source of new spacers in updated CRISPR arrays (Figure 1). Xue et al. (2016) show that conformational changes in Cascade correlate with the attenuation of interference and relative increase in adaptive priming (Figure 1).

FIGURE. Schematic of the conformational regulation of priming mechanisms.

Two states of Cascade exist in a dynamic equilibrium in which Cse1 is in an open (left) or closed (right) conformation. Targets containing PAM and protospacer variants that induce the closed conformation of the Cse1 subunit (left) recruit a nuclease-activated Cas3 that mediates target degradation. Cas3 degradation products form DNA duplexes enriched with NTT sequences in their 3’ overhang ends. These precursors are bound and processed by a Cas1-Cas2 complex and integrated as a new spacer (S-₀ in Red) into the CRISPR locus during primed spacer acquisition. Targets that favor the open conformation of Cse1 (left) block direct recruitment of Cas3 but still enable priming, likely through a Cas1-Cas2 dependent recruitment of a nuclease-inactivated Cas3. How the targets are generated for subsequent spacer acquisition is not known.

Examining escape mutants (containing a CTT-PAM and variant protospacers) that attenuate interference, Künne et al, (2016) showed that Cas3 degradation products serve as substrates for spacer acquisition by the Cas1-Cas2 integrase. Specifically, they demonstrate that during interference cleavage of plasmid DNA by Cas3 produces fragments of near spacer length that re-anneal to form duplexes, which likely contain both 5’ and 3’ overhangs. These fragments are also enriched for NTT sequences at their 3’ ends due to a preference of Cas3 to cleave thymine rich sequences (Figure 1). Thus, a fraction of the duplexes generated by Cas3 degradation are ideal substrates for the Cas1-Cas2 integrase, which preferentially binds DNA with 3’ overhangs containing a CTT sequence (Nuñez et al., 2015; Wang et al., 2015). By reconstituting spacer integration in vitro the authors confirmed that the Cas3 derived fragments can be bound and integrated into CRISPR arrays by the Cas1-Cas2 integrase, thus providing evidence for a priming mechanism that depends on the degradation of the target by nuclease-active Cas3, following its recruitment by Cascade.

During interference, the Cse1 subunit of Cascade recognizes PAM and directly recruits nuclease-active Cas3. Escape mutants with a PAM variant that blocks interference elicit an alternative response pathway in which nuclease-inactivated Cas3 is recruited through a mechanism dependent on Cas1 and Cas2. (Redding et al., 2015). In the available crystal structures, the Cse1 subunit adopts either a “closed” or “open” conformation and the “closed” conformation is proposed to be required for recruitment of Cas3 (Hayes et al., 2016). To determine if these conformations play a role in the switch between interference and priming Xue et al. (2016) established a bulk Forster Resonance Energy Transfer (FRET) system that enables measurement of conformational changes in Cse1 upon target binding. The authors measured FRET following binding of Cascade to DNA targets containing a canonical interference PAM (CTT), a priming PAM that attenuates interference and stimulates priming (TTT), and a priming PAM that blocks interference but stimulates priming (TCT). Based on this FRET, Cse1 adopts a “closed” conformation when bound to the CTT-PAM, an “open” conformation when bound the TCT-PAM, and a mixture of the two when bound to the TCT-PAM. The authors further show that mutations in Cse1 that destabilize the “closed” conformation significantly stimulate priming in cells. Thus, Cse1 exists in a dynamic equilibrium between “open” and “closed” conformations, and the extent to which the “open” conformation is favored correlates with the attenuation of interference and the relative increase in priming (Figure 1).

Overall, primed adaptation is a robust process that deals with escape mutants that can either partially or completely block interference. The studies discussed here suggest that when interference is attenuated, but not completely blocked, Cascade binds foreign DNA and Cse1 adopts the “closed” conformation (Xue et al. 2016). It then directly recruits nuclease-active Cas3, whose cleavage products serve as substrates for priming (Künne et al.). However, when Cascade binds to escape mutants that block interference, the Cse1 subunit will favor the “open” conformation. This likely precludes direct recruitment of nuclease-activated Cas3 (Xue et al. 2016) and instead leads to Cas1-Cas2-dependent recruitment of nuclease-inactivated Cas3 (Redding et al., 2015). In this case it is not clear if Cas3 or another nuclease will generate the substrates for priming. In summary, studies by Künne et al. (2016) and Xue et al. (2016) have provided new insights into the interplay between priming and interference, and established a framework for future studies to further delineate the underlying mechanisms of these complex pathways.