Cracking the Cas: Investigations into CRISPR RNA-guided bacterial immunity
- Author(s): Hochstrasser, Megan Lara
- Advisor(s): Doudna, Jennifer A
- et al.
In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)–
associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for
antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets
at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent
motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that
catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a
sequence matching that of the crRNA. Cascade comprises several Cas proteins and one
crRNA, forming a structure that binds and unwinds dsDNA to form an R-loop in which the
target strand of the DNA base pairs with the RNA guide sequence.
Single-particle electron microscopy reconstructions of dsDNA-bound Escherichia coli
Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of
the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding
that the DNA target and Cas3 co-localize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the
PAM region is unnecessary for target binding but critical for Cas3-mediated degradation.
In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for
Cas3 activation following target binding by Cascade. Together, these data show that the
CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA
target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.
Though the E. coli Cascade complex is well-studied, other Type I systems remain
relatively mysterious. We wondered how Desulfovibrio vulgaris Cascade/I-C could form
and function with just three distinct subunits, fewer than any other Type I or III interference
complex. Cas5c recognizes and cleaves the crRNA repeat via interactions with the 5′
handle region to which it remains associated. Cas5c binds to Cas7, which oligomerizes
cooperatively along the crRNA, capped by the crRNA stem-loop. Interactions with Cas5c,
Cas7, and the crRNA facilitate incorporation of Cas8c, a fusion of the large and small
subunits found in other systems. Subnanometer resolution cryo-electron microscopy
reconstructions of Cascade/I-C with and without target DNA reveal the dynamic role of
Cas8c, structural rearrangements for PAM recognition, the path of the displaced strand,
and a dramatic bend in the target duplex.