Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System

摘要

class="head no_bottom_margin" id="sec1title">IntroductionCRISPR-Cas is a set of microbial adaptive immune systems characterized by the insertion of short viral- or plasmid-derived DNA fragments called spacers into the CRISPR regions in the microbe’s chromosome. These spacers serve as an immunological memory and protect the host via Cas proteins.Fingerprinting invading DNA by inserting fragments as spacers into the CRISPR region is called adaptation (). Spacers serve as templates for generation of CRISPR RNA (crRNA) guides for the interference machinery encoded by Cas proteins. On the basis of the composition of ribonucleoprotein complexes that constitute the interference machinery, CRISPR-Cas systems are divided () into class 1 with multi-subunit interference complexes exemplified by Cascade () and class 2 with single-protein multi-domain effectors, such as Cas9 (, ) or Cas12 ().In a broad sense, the adaptation stage consists of the spacer capture step, where a prespacer is taken from the invading DNA, and the integration step, where a spacer is inserted into the CRISPR array. The prespacer acquired during the capture may require a separate step for end processing or may be processed directly in the integration complex (, , , , ). Despite the differences between different CRISPR-Cas systems, spacer integration is likely to be achieved in relatively similar ways, due to the fact that it is carried out by the highly conserved Cas1 and Cas2 proteins (, ).In a prototypical type I-E CRISPR-Cas system from E. coli, Cas1 and Cas2 form a complex that promotes spacer integration into the CRISPR array (, ). In this complex, a Cas2 dimer is sandwiched by two Cas1 dimers in a butterfly-like arrangement to provide a platform for the binding of a prespacer in the form of a 23-bp duplex with 5-nt, splayed or 3′ protruding ends (href="#bib35" rid="bib35" class=" bibr popnode">Nuñez et al., 2015b, href="#bib45" rid="bib45" class=" bibr popnode">Wang et al., 2015). A prespacer contains a protospacer (a sequence matching the spacer in the CRISPR array) and a PAM (protospacer adjacent motif) sequence, the latter being needed for the discrimination between self and non-self (href="#bib35" rid="bib35" class=" bibr popnode">Nuñez et al., 2015b, href="#bib45" rid="bib45" class=" bibr popnode">Wang et al., 2015). One of the Cas1 subunits recognizes the PAM sequence, which is subsequently removed by an as yet unknown mechanism, and a protospacer sequence is integrated into the CRISPR array (href="#bib35" rid="bib35" class=" bibr popnode">Nuñez et al., 2015b, href="#bib45" rid="bib45" class=" bibr popnode">Wang et al., 2015). The protospacer integration reaction proceeds by nucleophilic attack of both sides of the first repeat of the CRISPR region by the free 3′-hydroxyl ends of the protospacer. This reaction is carried out by the Cas1-Cas2 complex, with Cas1 playing the catalytic role in the integration reaction. For the type I-E system, integration specificity is enhanced by the integration host factor (IHF) binding to the leader sequence of the CRISPR region to impose a specific DNA structure that is recognized by the Cas1-Cas2 complex (href="#bib36" rid="bib36" class=" bibr popnode">Nuñez et al., 2016). The resulting product is repaired by host factors, and each integration event is accompanied by the duplication of the leader-proximal repeat sequence (href="#bib2" rid="bib2" class=" bibr popnode">Amitai and Sorek, 2016, href="#bib43" rid="bib43" class=" bibr popnode">Sternberg et al., 2016).The universal conservation of the Cas1 and Cas2 proteins implies that the spacer integration mechanism may share similarities across different CRISPR-Cas systems. However, the type II-A CRISPR-Cas systems of Streptococcus thermophilus and Streptococcus pyogenes encode four Cas proteins: Cas9; Cas1; Cas2; and Csn2 (href="/pmc/articles/PMC6620040/figure/fig1/" target="figure" class="fig-table-link figpopup" rid-figpopup="fig1" rid-ob="ob-fig1" co-legend-rid="lgnd_fig1">Figure 1A), all of which are essential for adaptation in vivo (href="#bib14" rid="bib14" class=" bibr popnode">Heler et al., 2015, href="#bib46" rid="bib46" class=" bibr popnode">Wei et al., 2015). However, although Cas9 nucleolytic activity is not required for protospacer integration, mutations of amino acid residues in Cas9, which are known to be involved in PAM recognition, lead to PAM-independent spacer acquisition, confirming that Cas9 is required for insertion of spacers with a correct PAM sequence (href="#bib14" rid="bib14" class=" bibr popnode">Heler et al., 2015, href="#bib46" rid="bib46" class=" bibr popnode">Wei et al., 2015). Although Csn2 is indispensable in the adaptation step, its function remains obscure. Previous studies revealed that the Csn2 protein is tetrameric and forms a toroidal structure (href="#bib3" rid="bib3" class=" bibr popnode">Arslan et al., 2013, href="#bib10" rid="bib10" class=" bibr popnode">Ellinger et al., 2012, href="#bib25" rid="bib25" class=" bibr popnode">Lee et al., 2012, href="#bib32" rid="bib32" class=" bibr popnode">Nam et al., 2011). Although no biochemical activity has been detected for Csn2, it has been shown to bind to double-stranded DNA ends (href="#bib10" rid="bib10" class=" bibr popnode">Ellinger et al., 2012, href="#bib25" rid="bib25" class=" bibr popnode">Lee et al., 2012, href="#bib32" rid="bib32" class=" bibr popnode">Nam et al., 2011). Recent work has shown that all four Cas proteins in this system interact with one another (href="#bib19" rid="bib19" class=" bibr popnode">Ka et al., 2018).href="/pmc/articles/PMC6620040/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">class="inline_block ts_canvas" href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=6620040_gr1.jpg" target="tileshopwindow">target="object" href="/pmc/articles/PMC6620040/figure/fig1/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC6620040/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">Figure 1Cas1, Cas2, Csn2, and Cas9 Proteins of CRISPR3-Cas System from S. thermophilus DGCC7710 Form a Stable Complex(A) Schematic representation of the CRISPR3-Cas locus.(B) CRISPR3-Cas system deletion mutants used to screen the complex assembly.(C) Results from purification of the complexes using the deletion mutants of the CRISPR3-Cas system depicted in (B). The CRISPR3-Cas system variants were co-expressed with Cas1 protein bearing a StrepII tag. The complexes were purified using StrepTrap column followed by size-exclusion chromatography and analyzed on SDS-PAGE. Cas1 and Cas2 form the core complex. Csn2 binds to Cas1-Cas2 complex (or vice versa), allowing binding of Cas9.(D) Cas1, Cas2, Csn2, and Cas9 proteins co-purify in the absence of crRNA and tracrRNA.(E) SDS-PAGE of the purified complex used for structural analysis.