CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites
November 14th, 2018
We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.
Overview
This article presents a genome engineering workflow for creating in vitro models of HIV-1 infection that accurately replicate proviral integration at specific genomic sites. The method utilizes CRISPR-Cas9 for site-specific genome manipulation, enabling targeted HIV-derived reporters.
Key Study Components
Area of Science
- Genome Engineering
- HIV-1 Research
- CRISPR-Cas9 Technology
Background
- Understanding HIV provirus integration is crucial for studying its effects on gene expression.
- In vitro models are essential for investigating HIV infection mechanisms.
- CRISPR/Cas9 allows for precise genomic modifications.
- Targeting specific integration sites can reveal insights into HIV biology.
Purpose of Study
- To develop in vitro models that mimic HIV-1 infection.
- To explore how proviral integration affects gene expression.
- To identify genomic sites selected during HIV infection.
Methods Used
- Utilization of UCSC Genome Browser for genomic sequence extraction.
- Selection of guide RNAs using the E-CRISP web tool.
- Targeting the Homo Sapiens Genome Reference Consortium Human Genome: Build 38.
- Generation and screening of single-cell clones for correct targeting.
Main Results
- Successful generation of in vitro models with targeted HIV-derived reporters.
- Verification of correct genomic targeting using CRISPR-Cas9.
- Insights into the influence of proviral integration on gene expression.
- Demonstration of the workflow by researchers in the lab.
Conclusions
- The developed workflow provides a robust method for studying HIV-1 integration.
- Targeted models can enhance understanding of HIV biology.
- This approach may lead to new therapeutic strategies against HIV.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables the generation of isogenic Jurkat reporter lines with HIV-derived sequences integrated at predefined genomic loci, providing a controlled system to study how proviral integration site influences viral gene expression and host cell responses. By modeling clinically relevant integration sites, the approach supports mechanistic de-risking in HIV target validation and preclinical assay development. The resulting clonal cell lines offer predictive value for evaluating latency-reversing agents and transcriptional regulators in a site-specific context.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of how specific genomic integration sites modulate HIV proviral transcription and host gene expression.
- Operational Value: Provides isogenic clonal lines that reduce biological variability in mechanistic studies of viral integration.
Screening & Assay Development
- Scientific Value: Generates standardized reporter systems for quantitative assessment of transcriptional activity under defined integration contexts.
- Operational Value: Supports assay reproducibility through clonal expansion and verification via flow cytometry, PCR, and Southern blotting.
Translational & Preclinical Research
- Scientific Value: Models HIV infection at selected loci to study site-dependent responses to latency-reversing compounds.
- Operational Value: Enables preclinical evaluation of transcriptional modulators in a genetically defined background.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target validation to preclinical screening, where site-specific reporter models inform lead identification and mechanistic de-risking.
- Discovery Biology: Facilitates hypothesis testing regarding integration site selection and its impact on viral transcriptional regulation.
- Screening: Delivers assay-ready clonal lines with verified reporter integration for compound response profiling.
- Analytics: Enables flow cytometry and PCR-based quantification of reporter expression as a functional readout of integration site effects.
- Translational Research: Connects integration site variability to differential responses in latency reversal assays.
- Enterprise Reuse: Establishes a reusable platform for generating site-specific HIV models across multiple loci and reporter constructs.
Operational & Enterprise Impact
- Scientific Value: Reduces confounding variables in HIV mechanism studies by controlling integration site.
- Operational Value: Standardizes model generation through CRISPR-Cas9-mediated homologous recombination and clonal validation.
- Strategic Value: Improves go/no-go decisions in antiviral development by linking integration site to transcriptional output.
- Portfolio Impact: Supports risk-adjusted prioritization of compounds based on site-specific efficacy profiles.
Implementation Considerations
- Requires expertise in CRISPR-Cas9 design, homology-directed repair, and clonal cell line screening.
- Dependent on access to flow cytometry, PCR, and Southern blotting for validation.
- Necessitates careful gRNA selection and off-target analysis using tools like E-CRISP and BLAST.
- Involves time-intensive single-cell cloning and expansion (2–3 months).
- Demands BSL3 containment if replication-competent reporters are used.
Biopharma FAQs
Chapters in this video
0:04
Title
0:42
Choice of Targeted Locus, Guide RNA (gRNA), and Targeting Vector Design
3:08
CRISPR-Cas9-based Targeting of Jurkat Cells
4:36
Generation of Clonal Lines and Screening for Correct Targeting
7:50
Screening of Single-cell Clones by Flow Cytometry and PCR
11:46
Results: Screening and Analysis of Single-cell Clones for Correct Reporter Integration
13:16
Conclusion
