Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3′ side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

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1Most type V CRISPR-Cas interference proteins use a single RuvC active site to make 2 RNA-guided breaks in double-stranded DNA substrates, an activity essential for both 3 bacterial immunity and genome editing applications. The best-studied of these 4 enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the 5 guide RNA displaces one of the DNA strands of a double-helical substrate, positioning 6 the DNase active site for first-strand cleavage. However, crystal structures and 7 biochemical data have not explained how the second strand is cut to complete the 8 double-strand break. Here, we show that Cas12a-mediated R-loop formation 9 destabilizes DNA at the second-strand cleavage site, which is located outside of the R-10 loop structure and beyond the 3′ end of the guide RNA. Chemical and fluorescent DNA 11 probes reveal that this destabilization is an intrinsic feature of DNA flanking the RNA-3′ 12 side of R-loops and does not require direct protein interactions. Interestingly, DNA 13 flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-14 loop structure may explain the uniformity of guide RNA architecture and the single-15 active-site cleavage mechanism that are fundamental features of all type V CRISPR-16Cas systems. 17 family occurs with opposing directionality (Fig.

Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposi’s sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus, deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is dependent upon a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we found that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV viral preinitiation complex and suggests that it functions as a complex to recognize late gene promoters. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. The release of infectious virions requires the production of capsid proteins and other late genes, whose production is transcriptionally controlled by a complex of six virally encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function five of its six components play in transcription. Here, we demonstrate that ORF66 is an essential component of this complex in KSHV and that its inclusion in the complex depends upon its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examined the assembly of the viral preinitiation complex at late gene promoters and found that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral preinitiation complex.

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