Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli

Abstract

Clusters of regularly interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes1. Type I account for 95% of CRISPR systems, and have been utilized to control gene expression and cell fate2,3. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defense) facilitates crRNA-guided invasion of double-stranded DNA (dsDNA) for complementary base-pairing with the target DNA strand, while displacing the non-target strand, forming an R-loop4,5. Cas3 nuclease/helicase is recruited subsequently to degrade two DNA strands4,6,7. Protospacer adjacent motif (PAM) flanking target DNA is crucial for self vs. foreign discrimination4,8–16. Here we present a 2.45 Å crystal structure of E. coli Cascade bound to a foreign dsDNA target. The 5′-ATG PAM is recognized in double-stranded form, from the minor groove side, by three structural features in Cse1. The promiscuity inherent to minor groove DNA recognition rationalizes the puzzling observation that a single Cascade can respond to several distinct PAM sequences. Optimal PAM recognition coincides with a wedge insertion, initiating the directional target DNA strand unwinding for segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind Cse2 dimer. These observations provide the structure basis for understanding the PAM-dependent directional R-loop formation process17,18.

CRISPR–Cas9 Structures and Mechanisms

“…This direct base readout through water-mediated hydrogen-bonding interactions in the major groove of the DNA confers greater sequence specificity and discrimination than that observed with the minor groove DNA recognition (83). Notably, Cascade also recognizes its PAM sequence in double-stranded form, but from the minor groove side, explaining the promiscuity of type I PAM recognition (29). In addition to base-specific contacts with GG dinucleotides, Cas9's CTD makes numerous hydrogen-bonding interactions with the deoxyribose-phosphate backbone of the PAM-containing nontarget DNA strand.…”

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

“…PAM recognition facilitates crRNA-guided strand invasion, allowing the guide sequence of the crRNA to hybridize to the complementary strand of the foreign DNA. Target binding induces a conformational change in the complex that displaces the noncomplementary strand of DNA, resulting in an R-loop structure (33)(34)(35)(36). The Cas3 nuclease-helicase is then recruited to degrade this displaced strand (11-13, 37, 38).…”

Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade

“…In the Type I–E CRISPR-Cas system of Escherichia coli , five stoichiometrically unequal proteins (Cse1 1 , Cse2 2 , Cas7 6 , Cas5e 1 , and Cas6e 1 ) assemble with a 61-nt crRNA to form the Cascade surveillance complex (Fig. 1A) (Brouns et al, 2008; Hayes et al, 2016; Jackson et al, 2014; Jore et al, 2011; Mulepati et al, 2014; Zhao et al, 2014). Similar to the Type II surveillance complex Cas9, Cascade searches for targets by first recognizing a short sequence called the protospacer-adjacent motif (PAM) (Mojica et al, 2009; Redding et al, 2015; Rollins et al, 2015; Sashital et al, 2012; Semenova et al, 2011; Sternberg et al, 2014).…”

“…Similar to the Type II surveillance complex Cas9, Cascade searches for targets by first recognizing a short sequence called the protospacer-adjacent motif (PAM) (Mojica et al, 2009; Redding et al, 2015; Rollins et al, 2015; Sashital et al, 2012; Semenova et al, 2011; Sternberg et al, 2014). PAM recognition destabilizes the DNA duplex by inserting a glutamine wedge located in the large Cse1 (Cas8e) subunit into the dsDNA adjacent to the PAM, enabling strand invasion and formation of an RNA-DNA heteroduplex in the seed region (positions 1–5 and 7–8 of the crRNA spacer) (Hayes et al, 2016; Xiao et al, 2017). Complementary base pairing in the seed region efficiently promotes further R-loop formation (Semenova et al, 2011; Szczelkun et al, 2014).…”