Degradation of Phage Transcripts by CRISPR-Associated RNases Enables Type III CRISPR-Cas Immunity.

Type III-A CRISPR-Cas systems defend prokaryotes against viral infection using CRISPR RNA (crRNA)-guided nucleases that perform co-transcriptional cleavage of the viral target DNA and its transcripts. Whereas DNA cleavage is essential for immunity, the function of RNA targeting is unknown. Here, we show that transcription-dependent targeting results in a sharp increase of viral genomes in the host cell when the target is located in a late-expressed phage gene. In this targeting condition, mutations in the active sites of the type III-A RNases Csm3 and Csm6 lead to the accumulation of the target phage mRNA and abrogate immunity. Csm6 is also required to provide defense in the presence of mutated phage targets, when DNA cleavage efficiency is reduced. Our results show that the degradation of phage transcripts by CRISPR-associated RNases ensures robust immunity in situations that lead to a slow clearance of the target DNA.

Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity.

Immune systems must recognize and destroy different pathogens that threaten the host. CRISPR-Cas immune systems protect prokaryotes from viral and plasmid infection utilizing small CRISPR RNAs that are complementary to the invader's genome and specify the targets of RNA-guided Cas nucleases. Type III CRISPR-Cas immunity requires target transcription, and whereas genetic studies demonstrated DNA targeting, in vitro data have shown crRNA-guided RNA cleavage. The molecular mechanism behind these disparate activities is not known. Here, we show that transcription across the targets of the Staphylococcus epidermidis type III-A CRISPR-Cas system results in the cleavage of the target DNA and its transcripts, mediated by independent active sites within the Cas10-Csm ribonucleoprotein effector complex. Immunity against plasmids and DNA viruses requires DNA, but not RNA, cleavage activity. Our studies reveal a highly versatile mechanism of CRISPR immunity that can defend microorganisms against diverse DNA and RNA invaders.

Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.

Genetic characterization of antiplasmid immunity through a type III-A CRISPR-Cas system.

Many prokaryotes possess an adaptive immune system encoded by clustered regularly interspaced short palindromic repeats (CRISPRs). CRISPR loci produce small guide RNAs (crRNAs) that, in conjunction with flanking CRISPR-associated (cas) genes, combat viruses and block plasmid transfer by an antisense targeting mechanism. CRISPR-Cas systems have been classified into three types (I to III) that employ distinct mechanisms of crRNA biogenesis and targeting. The type III-A system in Staphylococcus epidermidis RP62a blocks the transfer of staphylococcal conjugative plasmids and harbors nine cas-csm genes. Previous biochemical analysis indicated that Cas10, Csm2, Csm3, Csm4, and Csm5 form a crRNA-containing ribonucleoprotein complex; however, the roles of these genes toward antiplasmid targeting remain unknown. Here, we determined the cas-csm genes that are required for antiplasmid immunity and used genetic and biochemical analyses to investigate the functions of predicted motifs and domains within these genes. We found that many mutations affected immunity by impacting the formation of the Cas10-Csm complex or crRNA biogenesis. Surprisingly, mutations in the predicted nuclease domains of the members of the Cas10-Csm complex had no detectable effect on antiplasmid immunity or crRNA biogenesis. In contrast, the deletion of csm6 and mutations in the cas10 Palm polymerase domain prevented CRISPR immunity without affecting either complex formation or crRNA production, suggesting their involvement in target destruction. By delineating the genetic requirements of this system, our findings further contribute to the mechanistic understanding of type III CRISPR-Cas systems.

A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs.

Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.

Kinetic analysis of DNA strand joining by Chlorella virus DNA ligase and the role of nucleotidyltransferase motif VI in ligase adenylylation.

Chlorella virus DNA ligase (ChVLig) is an instructive model for mechanistic studies of the ATP-dependent DNA ligase family. ChVLig seals 3'-OH and 5'-PO(4) termini via three chemical steps: 1) ligase attacks the ATP α phosphorus to release PP(i) and form a covalent ligase-adenylate intermediate; 2) AMP is transferred to the nick 5'-phosphate to form DNA-adenylate; 3) the 3'-OH of the nick attacks DNA-adenylate to join the polynucleotides and release AMP. Each chemical step requires Mg(2+). Kinetic analysis of nick sealing by ChVLig-AMP revealed that the rate constant for phosphodiester synthesis (k(step3) = 25 s(-1)) exceeds that for DNA adenylylation (k(step2) = 2.4 s(-1)) and that Mg(2+) binds with similar affinity during step 2 (K(d) = 0.77 mM) and step 3 (K(d) = 0.87 mM). The rates of DNA adenylylation and phosphodiester synthesis respond differently to pH, such that step 3 becomes rate-limiting at pH ≤ 6.5. The pH profiles suggest involvement of one and two protonation-sensitive functional groups in catalysis of steps 2 and 3, respectively. We suggest that the 5'-phosphate of the nick is the relevant protonation-sensitive moiety and that a dianionic 5'-phosphate is necessary for productive step 2 catalysis. Motif VI, located at the C terminus of the OB-fold domain of ChVLig, is a conserved feature of ATP-dependent DNA ligases and GTP-dependent mRNA capping enzymes. Presteady state and burst kinetic analysis of the effects of deletion and missense mutations highlight the catalytic contributions of ChVLig motif VI, especially the Asp-297 carboxylate, exclusively during the ligase adenylylation step.

Functional dissection of the DNA interface of the nucleotidyltransferase domain of chlorella virus DNA ligase.

Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive ß-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3'-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3'-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3'-OH nucleoside in the catalysis of DNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig.

Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase.

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.

Genome DNA leakage of Adeno-Associated virus under freeze-thaw stress.

Adeno-associated virus (AAV) has become an emerging tool for human gene therapies. Currently, AAV gene therapies are subjected to multiple freeze-thaw cycles during manufacturing, storage, transportation, and administration. While studies have shown that multiple freeze-thaw cycles led to a decrease in transduction efficiency, the AAV degradation mechanism during freeze-thaw is not well understood. Here, we have characterized the impact of freeze-thaw on AAV8 by employing a variety of assays, which revealed significant increases in the amount of free single-stranded DNA (ssDNA) in AAV8 formulations after multiple freeze-thaw cycles. Subsequent analysis using Next Generation Sequencing (NGS) revealed that the ssDNA primarily consisted of genome DNA, indicating that the increased ssDNA leaked out from AAV8. Experiments performed using different serotypes of AAV confirmed the pervasiveness of such behavior amongst AAVs. In addition, formulation screening studies were performed to understand the impact on genome DNA leakage from AAV. The formulation screening results showed that the addition of 10% sucrose and 0.1% poloxamer 188 to Dulbecco's phosphate-buffered saline (DPBS) reduced the leakage of ssDNA in AAV samples after freeze-thaw cycles compared to the base formulation of DPBS alone. These findings shed new light on the degradation mechanism of AAVs and stabilization of the AAV-based gene therapies.

Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris.

CRISPRs (clustered regularly interspaced short palindromic repeats) provide bacteria and archaea with RNA-guided acquired immunity to invasive DNAs. CRISPR-associated (Cas) proteins carry out the immune effector functions. Cas2 is a universal component of the CRISPR system. Here, a 1.35 Šresolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. DvuCas2 is a homodimer, with each protomer consisting of an N-terminal ßαßßαß ferredoxin fold (amino acids 1-78) to which is appended a C-terminal segment (amino acids 79-102) that includes a short 3(10)-helix and a fifth ß-strand. The ß5 strands align with the ß4 strands of the opposite protomers, resulting in two five-stranded antiparallel ß-sheets that form a sandwich at the dimer interface. The DvuCas2 dimer is stabilized by a distinctive network of hydrophilic cross-protomer side-chain interactions.

Incomplete prophage tolerance by type III-A CRISPR-Cas systems reduces the fitness of lysogenic hosts.

CRISPR-Cas systems offer an immune mechanism through which prokaryotic hosts can acquire heritable resistance to genetic parasites, including temperate phages. Co-transcriptional DNA and RNA targeting by type III-A CRISPR-Cas systems restricts temperate phage lytic infections while allowing lysogenic infections to be tolerated under conditions where the prophage targets are transcriptionally repressed. However, long-term consequences of this phenomenon have not been explored. Here we show that maintenance of conditionally tolerant type III-A systems can produce fitness costs within populations of Staphylococcus aureus lysogens. The fitness costs depend on the activity of prophage-internal promoters and type III-A Cas nucleases implicated in targeting, can be more severe in double lysogens, and are alleviated by spacer-target mismatches which do not abrogate immunity during the lytic cycle. These findings suggest that persistence of type III-A systems that target endogenous prophages could be enhanced by spacer-target mismatches, particularly among populations that are prone to polylysogenization.