Engineering, decoding and systems-level characterization of chimpanzee cytomegalovirus.

The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious viruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci and a deletion-prone region within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the viral transcriptome and proteome and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV's cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification.

CRISPR base editing screens enable analysis of disease-associated variants at scale; however, variable efficiency and precision confounds the assessment of variant-induced phenotypes. Here, we provide an integrated experimental and computational pipeline that improves estimation of variant effects in base editing screens. We use a reporter construct to measure guide RNA (gRNA) editing outcomes alongside their phenotypic consequences and introduce base editor screen analysis with activity normalization (BEAN), a Bayesian network that uses per-guide editing outcomes provided by the reporter and target site chromatin accessibility to estimate variant impacts. BEAN outperforms existing tools in variant effect quantification. We use BEAN to pinpoint common regulatory variants that alter low-density lipoprotein (LDL) uptake, implicating previously unreported genes. Additionally, through saturation base editing of LDLR, we accurately quantify missense variant pathogenicity that is consistent with measurements in UK Biobank patients and identify underlying structural mechanisms. This work provides a widely applicable approach to improve the power of base editing screens for disease-associated variant characterization.

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification.

CRISPR base editing screens are powerful tools for studying disease-associated variants at scale. However, the efficiency and precision of base editing perturbations vary, confounding the assessment of variant-induced phenotypic effects. Here, we provide an integrated pipeline that improves the estimation of variant impact in base editing screens. We perform high-throughput ABE8e-SpRY base editing screens with an integrated reporter construct to measure the editing efficiency and outcomes of each gRNA alongside their phenotypic consequences. We introduce BEAN, a Bayesian network that accounts for per-guide editing outcomes and target site chromatin accessibility to estimate variant impacts. We show this pipeline attains superior performance compared to existing tools in variant classification and effect size quantification. We use BEAN to pinpoint common variants that alter LDL uptake, implicating novel genes. Additionally, through saturation base editing of LDLR, we enable accurate quantitative prediction of the effects of missense variants on LDL-C levels, which aligns with measurements in UK Biobank individuals, and identify structural mechanisms underlying variant pathogenicity. This work provides a widely applicable approach to improve the power of base editor screens for disease-associated variant characterization.

Emerging Mechanisms of G1/S Cell Cycle Control by Human and Mouse Cytomegaloviruses.

Cytomegaloviruses (CMVs) are among the largest pathogenic viruses in mammals. To enable replication of their long double-stranded DNA genomes, CMVs induce profound changes in cell cycle regulation. A hallmark of CMV cell cycle control is the establishment of an unusual cell cycle arrest at the G1/S transition, which is characterized by the coexistence of cell cycle stimulatory and inhibitory activities. While CMVs interfere with cellular DNA synthesis and cell division, they activate S-phase-specific gene expression and nucleotide metabolism. This is facilitated by a set of CMV gene products that target master regulators of G1/S progression such as cyclin E and A kinases, Rb-E2F transcription factors, p53-p21 checkpoint proteins, the APC/C ubiquitin ligase, and the nucleotide hydrolase SAMHD1. While the major themes of cell cycle regulation are well conserved between human and murine CMVs (HCMV and MCMV), there are considerable differences at the level of viral cell cycle effectors and their mechanisms of action. Furthermore, both viruses have evolved unique mechanisms to sense the host cell cycle state and modulate the infection program accordingly. This review provides an overview of conserved and divergent features of G1/S control by MCMV and HCMV.

Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease.

Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent protein patterns identified the recombination status of individual cells. In derivatives of a CHO master cell line the expression of the introduced transgene of interest could be dramatically increased almost 20-fold by subsequent deletion of the fluorescent protein gene that provided the initial isolation principle. The same master cell line was employed in a comparative analysis using CRISPR/Cas9 for transgene integration in identical loci. Even though the overall targeting efficacy was comparable, multi-loci targeting was considerably more effective for Cas9-mediated transgene insertion when compared to RMCE. While Cas9 is inherently more flexible, our results also alert to the risk of aberrant recombination events around the cut site. Together, this study points at the individual strengths in performance of both systems and provides guidance for their appropriate use.