Coding and non-coding elements comprise a regulatory network controlling transcription in Kaposi’s sarcoma-associated herpesvirus

Question

1. What are the potential regulatory elements in DNA viruses?

2. What is the purpose of using CRISPRi tiling in identifying regulatory regions in the KSHV genome?

3. How does CRISPRi recruitment affect viral genes locally?

4. How can CRISPRi alone distinguish regulatory regions controlling ORF68 expression?

Accepted Answer

Potential regulatory elements in DNA viruses include enhancer sequences, tRNA-like elements, miRNAs, origin RNAs, circular RNAs, and long ncRNAs. Enhancer sequences, such as the first described enhancer on SV40, the EIIA enhancer on adenovirus, and the major immediate enhancer element in the betaherpesvirus human cytomegalovirus, play a crucial role in viral gene transcription. tRNA-like elements, like those found in the gammaherpesvirus MHV68, control latency and egress. miRNAs, origin RNAs, circular RNAs, and long ncRNAs also contribute to the regulation of viral gene expression. These elements are essential for the lifecycle of DNA viruses and understanding their regulatory mechanisms can provide insights into viral pathogenesis and potential therapeutic targets.

Accepted Answer

The purpose of using CRISPRi tiling in identifying regulatory regions in the KSHV genome is to test the effect of each viral locus on a protein reporter of ORF68, an early gene involved in packaging. By delivering a library of guide RNAs densely tiling the KSHV genome, researchers can identify regions of the viral genome that affect ORF68 expression. This method allows for the identification of regulatory features and the effects of suppressing ORF68 expression early in the viral life cycle. The technique also helps in understanding the transcriptional effects of CRISPRi at each regulatory region on the viral genome.

Accepted Answer

CRISPRi recruitment to the viral genome inhibits many genes locally. In the study, polyA+ RNA-seq was performed at 24 hours post-reactivation on the previously validated guide pools. The results showed that CRISPRi targeting specific loci, such as ORF50, TSS75, and TSS57, caused a dramatic downregulation of nearly all viral genes. This downregulation aligns with the critical roles of these proteins in the viral lifecycle. ORF50 is a master regulator of KSHV lytic reactivation, ORF75 is required to prevent innate immune suppression of viral gene expression, and ORF57 has various viral functions, including the export of viral mRNA from the nucleus. In contrast, guides targeting TSS68 and TSSalt had a limited global effect, only strongly inhibiting a small number of genes each. The strongest downregulated genes were found within the local region of the guides, suggesting that CRISPRi inhibits transcription in a region of approximately 2.5 kb around the targeting site. This effect was observed even when CRISPRi increased ORF68 mRNA expression, indicating both local and global effects of CRISPRi at the target loci.

Accepted Answer

CRISPRi alone is unable to distinguish how regulatory regions may act to control ORF68 expression, whether by preventing protein-coding transcription of a regulator or by impeding a non-coding mechanism. To overcome these limitations, a CRISPR nuclease tiling screen was performed in the same HaloTag-ORF68 reporter line. The screen identified coding regions underlying each regulatory locus, such as ORF50, HygroR, ORF68, and ORF75, which control ORF68 expression at 24h postreactivation. Disruption of these coding sequences had similar effects on ORF68 expression as CRISPRi repression at their corresponding loci. However, for other CRISPRi loci, no corresponding effect of disrupting coding regions was observed. This suggests that CRISPRi alone cannot distinguish between coding and non-coding regulatory regions controlling ORF68 expression.

Accepted Answer

Noncoding elements may act indirectly, affecting the expression of viral proteins. By analyzing RNA-seq data, researchers found correlations between regulatory regions and their targets. TSSalt and polyA75 showed strong correlations with ORF68 and ORF75 expression, respectively. TSS72, however, had a strong anticorrelation with polyA75 and TSS75, suggesting it represses ORF75 expression. Weak correlations were observed for TSS57. These findings, along with data from Figure 3, helped create a model regulatory network controlling ORF68 expression. The network components were then tested for their impact on virion production in KSHV-infected cells. Targeting coding elements reduced virion production, while targeting TSS72 or TSSalt did not. This suggests that while ORF68 expression is essential late in infection, it may be dispensable at early time points.

Accepted Answer

CRISPRi on the viral genome effectively suppresses gene expression in a ~2-5 kb local region. In the densely encoded herpesviral genome, this footprint covers multiple elements, any of which could be responsible for the observed regulatory activity. A complementary Cas9 nuclease screen was used to disrupt the coding elements in these regions, revealing that for many of these regions their activity must be explained by non-coding elements that cannot be disrupted by Cas9. RNA expression patterns were used to predict functional targets of these elements, presenting a complete model for viral regulation of ORF68 and demonstrating that these regulatory elements play key roles in the completion of the KSHV lifecycle.

Accepted Answer

In CRISPRi screening, ORF68 expression is analyzed by reactivating cells with specific reactivation agents, sorting high and low expression cells, unfixing cells, extracting DNA, amplifying the sgRNA locus, ligating adaptors, sequencing libraries, and calculating enrichment scores. A 500 bp sliding window is used to identify significant peaks by comparing guide enrichment and negative control enrichment scores using a Mann-Whitney test. An arbitrary p-value cutoff is applied to identify peaks.

Accepted Answer

For each pool of sgRNAs, independent lentiviruses are produced and then pooled. These pooled lentiviruses are applied to CRISPRi-positive HaloTag-ORF68 iSLK cells for 48 hours. This process allows for the delivery of sgRNAs to the target cells, enabling the study of gene expression and protein analysis in the context of CRISPR interference (CRISPRi). The application of pooled lentiviruses ensures a consistent and efficient delivery of sgRNAs to the cells, facilitating the investigation of gene regulation and protein characterization in the research study.

Accepted Answer

Library quality was assessed on an AATI (now Agilent) Fragment Analyzer. This instrument measures the size distribution of the library fragments, ensuring that the library preparation process was successful and the library is suitable for sequencing. The Fragment Analyzer provides valuable information about the library's quality, including the presence of any degraded or contaminated fragments. By assessing the library quality, researchers can ensure that the sequencing data obtained will be reliable and accurate, leading to more meaningful results in their analysis.

Accepted Answer

The Cas9 screen was performed using a Cas9+ iSLK cell line infected with a copy of BAC16 containing a HaloTag-ORF68 fusion. The library was amplified using staggered primers and a modified amplification protocol previously described. qPCR was used to determine cycle number at 1/4 CT. All PCR products were run over a single Minelute column (Qiagen) for each sample. Size selection was performed using a gel extraction (Thermo). The library was sequenced on Illumina NextSeq 2000 with a 150bp single-read using Illumina sequencing primers. Adaptors were removed using cutadapt, and reads were aligned to the library using bowtie. Log2 values were calculated, and enrichment values were averaged from two replicates. For each exon boundary, a window of 500 bp on one side was tested against a 500 bp on the other to calculate p-values using MW test. Median values were used to determine the sign of the shift. Exons with p values < 0.001 and consistent signs were considered hits.

Coding and non-coding elements comprise a regulatory network controlling transcription in Kaposi’s sarcoma-associated herpesvirus

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Most frequently asked questions

1. What are the potential regulatory elements in DNA viruses?

2. What is the purpose of using CRISPRi tiling in identifying regulatory regions in the KSHV genome?

3. How does CRISPRi recruitment affect viral genes locally?

4. How can CRISPRi alone distinguish regulatory regions controlling ORF68 expression?

5. How do noncoding elements impact CRISPRi peaks?

6. What regulatory events were identified across the KSHV genome using CRISPRi and a tiling library?

7. How is ORF68 expression analyzed in CRISPRi screening?

8. How are sgRNAs pooled and applied to CRISPRi-positive cells?

9. How was library quality assessed?

10. How was the Cas9 screen performed in the CRISPR nuclease screen and analysis section?

Citations

Advances in computational and experimental approaches for deciphering transcriptional regulatory networks: Understanding the roles of cis-regulatory elements is essential, and recent research utilizing MPRAs, STARR-seq, CRISPR-Cas9, and machine learning has yielded valuable insights.

References

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Ultrafast and memory-efficient alignment of short DNA sequences to the human genome

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The Ubiquitin Code

Ancient DNA and the rewriting of human history: be sparing with Occam's razor.

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