Skip to main content
eScholarship
Open Access Publications from the University of California

UC Berkeley

UC Berkeley Electronic Theses and Dissertations bannerUC Berkeley

Cracking the Cas: Investigations into CRISPR RNA-guided bacterial immunity

Abstract

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.

Main Content
For improved accessibility of PDF content, download the file to your device.
Current View