The first pooled, genome-wide CRISPR-Cas9 knockout (KO) screen with authentic HBV infection is performed and these results are integrated with two orthogonal pooled screens to identify host factors to expand the catalog of HBV host factors and provide a scalable framework for host target discovery.
Abstract
Hepatitis B virus (HBV) chronically infects approximately 250 million people worldwide, and reliable curative therapies are lacking. A broader understanding of viral-host interactions could accelerate efforts to find new host-centric therapeutic targets. However, inefficient cell culture systems and limited replication markers compatible with pooled screening have precluded the widespread use of genetic perturbation screens. Here, we performed the first pooled, genome-wide CRISPR-Cas9 knockout (KO) screen with authentic HBV infection and integrated these results with two orthogonal pooled screens to identify host factors. We selected 72 genes for a multi-step assessment that included arrayed validation assays using both HBV infection and pgRNA transfection. We then independently tested thirteen genes using high-efficiency bulk KO experiments to guide further investigations of both antiviral and proviral factors. In both KO and siRNA-mediated knockdown experiments, depletion of the top antiviral factor, EXOC1, enhanced multiple HBV replication markers, and transcriptomic analysis revealed activation of hypoxia- and HIF-1 gene signatures. Three proviral factors, IRF2, WDR48, and ZCCHC14, were investigated in vivo using a human liver chimeric mouse model, which demonstrated that ZCCHC14 KO greatly reduced HBV replication and spread. Together, these complementary in vitro and in vivo platforms expand the catalog of HBV host factors and provide a scalable framework for host target discovery.
Lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH) are reported, a system that enables PHH expansion in liver-humanized mice and enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.
Ansgar F. Stenzel, Antonis Athanasiadis, Georgios Dangas et al.· bioRxiv· 0 citations
Viral genome diversity may limit the effectiveness of antiviral RNA-editing tools such as CRISPR-Cas13 that can be used to destroy specific mRNA targets, by introducing mismatches between viral RNA targets and CRISPR guide RNAs (crRNAs). These mismatches can reduce target recognition and cleavage efficiency, diminishing antiviral activity and increasing the risk of viral escape. The extent to which natural viral genomic variability limits CRISPR-Cas13 efficacy remains unclear. Here, we used hepatitis B virus (HBV), which has substantial genetic diversity, as a model to assess the impact of viral genome variation on Cas13b activity in vitro. The efficacy of PspCas13b was examined across six HBV genotypes and sub-genotypes using five crRNAs that had up to five mismatches to the target region. We showed that crRNAs with one mismatch to the target strongly suppressed viral antigen expression for all genotypes tested, while some crRNAs with three or more mismatches were less effective. Restoring complementarity using spacer-target mutagenesis improved the level of knockdown for some but not all HBV genotypes, suggesting that sequence specificity alone did not control PspCas13b efficacy. Our findings show that a “one size fits all” approach for PspCas13b-mediated treatment of HBV is unlikely to be effective, but the impact of sequence variability on PspCas13b efficacy can be readily addressed through appropriate design of crRNAs. This approach will likely be necessary for all viral pathogens with highly variant genomes. IMPORTANCE CRISPR-Cas13 is being explored as a novel antiviral for several viral infections. Viral sequence divergence can compromise CRISPR-Cas13 efficacy by introducing mismatches between therapeutic guide RNAs and viral targets. However, the impact of naturally occurring viral genomic variation on CRISPR-Cas13 efficacy remains poorly understood. Using hepatitis B virus (HBV) as a model, we showed that the effect of mismatches on Cas13b efficacy was context-dependent and varied for different crRNAs, HBV genotypes and target sites. Restoring complementarity improved the efficacy for some, but not all crRNAs, suggesting that Cas13b efficacy was not solely influenced by the number of mismatches. As the target sequence may differ between viral variants, this study advances our understanding of the impact of mismatches on Cas13b efficacy and provides further insights into using Cas13b as a novel antiviral.
Mai Anh Thu Le, L. McCoullough, Zak T. Janetzki et al.· bioRxiv· 0 citations
This review systematically summarize recent progress in CRISPR/Cas9-based screening studies of major livestock and poultry viruses, including foot-and-mouth disease virus (FMDV), swine enteric coronaviruses, African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), avian leukosis virus (ALV), and other zoonotic pathogens.
A virus-like particle (VLP)-based toolkit that delivers diverse CRISPR editing modalities to human monocytes, macrophages and dendritic cells with high efficiency while preserving viability and innate immune responsiveness is presented.
Hyuncheol Jung, Pascal Devant, Carter Ching et al.· Nature Biotechnology· 0 citations
Background: Leukemia is a cancer of hematopoietic stem cells in the bone marrow. It is classified as lymphoid or myeloid, and as acute (rapid onset) or chronic (slow progression). Advances in CRISPR technology enable deeper study of leukemia biology and therapeutic targets. While AML, CML, ALL, and CLL are distinct, comparative studies highlight shared and unique features. Identifying key pathways across subtypes may yield improved treatments. Our lab has established a unique CRISPR Activation (CRISPRa) platform for gain-of-function screening applications. We hypothesize that genome-wide CRISPRa screening will uncover subtype-specific genetic dependencies and drug resistance mechanisms, offering novel therapeutic insights.
We engineered leukemia cell lines by nucleofecting a self-selecting CRISPRa PiggyBac plasmid carrying a blasticidin resistance gene, enabling selection of CRISPRa-competent cells. Functionality was validated using lentiviral delivery of sgRNAs targeting cell surface markers. Cells were transduced with our whole-genome CRISPRa library, Sonata, at MOI 0.4, followed by puromycin selection to enrich for sgRNA-expressing cells. Post-transduction, cells were harvested at various timepoints for sequencing to track sgRNA abundance and identify growth-modulating genes.
We engineered and validated four CRISPRa-competent leukemia lines: K562 (CML), Jurkat (T-ALL), THP-1 (AML), and HL60 (AML). We have completed whole-genome screening campaigns in the CML and T-ALL backgrounds, identifying hundreds of shared and context specific growth modifiers. Functional validation, pathway analysis, and potential clinical significance is ongoing, as is expansion of our screens with the AML contexts.
Conclusions: Our screens reveal genes that, upon activation, influence leukemia cell growth. These findings support discovery of new therapeutic targets and enhance understanding of leukemia subtype biology, guiding future personalized treatment strategies.
The Cole Foundation, FHMR
Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Jeffrey Sullivan, Xiaozen Wen, Gabriela Flores-Vargas et al.· Journal of Immunology· 0 citations
Summary Influenza A virus (IAV) remains a major threat to human and animal health, while the emergence of drug-resistant strains necessitates new antiviral strategies. Here, we developed an integrative host-directed drug discovery framework combining functional genomics and pharmacotranscriptomics. By aggregating published genome-wide screens, we assigned host genes functional scores reflecting their effects on IAV replication and used these scores to estimate the antiviral status of host cells. Screening nearly 20,000 drug-induced transcriptional signatures identified compounds that shift host gene expression toward an antiviral state. Among 54 selected hits, 18 showed anti-IAV activity. Notably, lithocholic acid and ALW-II-49-7 inhibited viral replication in vitro and protected mice from lethal infection in vivo. This host-targeted framework provides a systematic and scalable strategy for discovering antivirals that are less susceptible to resistance and potentially applicable to other rapidly evolving pathogens.
Jianfa Qiu, Xuecong Xing, Jing-Feng Wang et al.· iScience· 0 citations
