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 the CasA-E 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 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound 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 colocalize 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.

Summary CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease-helicase for target degradation. Here we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways, dictated by the presence or absence of a protospacer adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short singlestranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent upon the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes, and supports a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nucleasedead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA-target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9-DNA interaction and broaden the toolkit for investigating off-target binding behavior.DNA | molecular biophysics | kinetics | sequencing | CRISPR C RISPR-associated protein 9 (Cas9) is programmed to bind its target DNA by a guide RNA (gRNA) that, once loaded, forms a ribonucleoprotein (RNP) complex. The Streptococcus pyogenes CRISPR system, the most extensively studied and applied system to date, targets a 23-bp DNA sequence containing (i) an "NGG" protospacer adjacent motif (PAM) element downstream of the single-guide RNA (sgRNA) target DNA (1) and (ii) a 20-bp sequence upstream of the PAM bearing complementarity to the gRNA (2). Genome engineering applications leverage the nuclease activity of the Cas9 RNP, but Cas9 engineered to lack the residues required for cleavage [dCas9 (nuclease-dead Cas9)] has proven valuable by enabling the creation of customizable and programmable DNA binding elements that can activate and repress gene expression with high precision (CRISPRa and CRISPRi) (3).The biophysical underpinnings of the Cas9 target search have been investigated both by directed biochemical assays (4, 5) and through measurements of off-target Cas9 activity (6-11). These studies have led to a model for binding wherein Cas9 proceeds through a series of steps starting with PAM recognition, followed by DNA melting, RNA strand invasion, and heteroduplex formation dependent on complementarity with a 5-10-bp seed. Structural data have further suggested that conformational changes in the HNH domain reposition catalytic residues and permit allosteric regulation of the RuvC domain. This conformational gating ensures that cleavage occurs only in the context of substantial homology between gRNA and target (12, 13).The specificity of Cas9 DNA binding is crucial for all potential applications of Cas9's RNA-programmable targeting. Localization of dCas9...

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The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ∼75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest. IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a “do-it-yourself” kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.