CRISPRa Screening for Influenza Host Factors
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Solution Overview
Problem
Current methods for identifying host factors regulating influenza virus infection are limited, as they primarily rely on loss-of-function screens that fail to detect genes essential for cell viability or those with redundant functions, leaving unexplored genetic space and potentially missing new classes of viral co-factors.
Innovation Solution
Employing CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi) systems to programmatically modulate host gene expression using the influenza virus-encoded Cas9, allowing for gain- and loss-of-function screens by targeting specific genomic loci with CRISPR-Cas9 technology, thereby identifying host factors that enhance or inhibit viral replication.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If loss-of-function screens are used to identify host factors, then genes already expressed in the system can be probed, but genes essential for cell viability, genes with redundant functions, or gene products needed in limited quantities cannot be detected
Solution Approach 1:
The patent inverts the traditional loss-of-function screening approach by implementing gain-of-function screening using CRISPRa technology. Instead of knocking out genes to observe effects, the system activates gene expression to identify pro-viral host factors. This inversion enables detection of genes that were previously undetectable by loss-of-function methods, including essential genes and those with redundant functions.
Solution Approach 2:
The patent introduces an intermediary mechanism (CRISPRa system with dCas9-VP64 fusion protein and sgRNAs) that mediates gene activation. This intermediary allows selective upregulation of host genes during viral infection, enabling the identification of host factors that enhance viral replication without requiring gene knockout or permanent cellular modification.
2Productivity
If CRISPR activation system is implemented using viral-encoded Cas9, then host gene expression can be modulated during infection, but the construct remains inactive until viral transcription begins
Solution Approach 1:
The patent implements preliminary action by pre-assembling the CRISPRa machinery (dCas9-VP64 fusion protein and sgRNA expression constructs) within the viral genome before infection. The system remains dormant during viral entry and only becomes active after viral transcription initiates, ensuring that gene activation occurs at the optimal time during the infection cycle when host factors are most needed for viral replication.
Solution Approach 2:
The patent creates a dynamic system where the CRISPRa construct transitions from an inactive state during viral entry to an active state during viral transcription. The system dynamically responds to the infection timeline, activating gene expression only when viral RNA polymerase begins transcribing the viral genome, thereby coordinating host gene modulation with viral replication needs.
3Measurement precision
If a library of 70,000 sequences is prepared to blanket the genome, then comprehensive coverage of human genes is achieved, but the complexity of library construction and sequencing analysis increases
Solution Approach 1:
The patent segments the comprehensive genome-wide screening into manageable components: the viral genome is divided to accommodate multiple sgRNA expression cassettes, each targeting specific host genes. The library of 70,000 sequences is organized as modular sgRNA units that can be independently synthesized and assembled into the viral construct, simplifying the overall library construction process while maintaining comprehensive genome coverage.
Solution Approach 2:
The patent uses deep sequencing to create digital copies of the viral population, allowing comprehensive analysis of sgRNA enrichment patterns without physically manipulating the entire library. By sequencing viral RNA from infected cells, the system generates accurate copies of the sgRNA population distribution, enabling precise measurement of host factor importance while avoiding the complexity of physical library management.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables the identification of host regulators that significantly impact influenza virus replication, with some factors increasing viral yields up to 10-fold, and provides a method to screen for compounds that alter pathogen activity, potentially leading to new therapeutic strategies.
Implementation Method 1
CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi). In those methods, the sequence of a single guide RNA ('sgRNA') directs Cas9 to a specific location, and the catalytically inactive Cas9 has been modified to recruit transcriptional activators or repressors to modify gene expression at that location.
Implementation Method 2
CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi). In those methods, the sequence of a single guide RNA ('sgRNA') directs Cas9 to a specific location, and the catalytically inactive Cas9 has been modified to recruit transcriptional activators or repressors to modify gene expression at that location.
Implementation Method 3
An influenza virus was used to express the CRISPR sgRNA, in a technique referred to as transcriptional regulation by pathogen-programmed Cas9 (TRPPC). This way, the construct is inactive until after a virus infects a host cell and begins to be transcribed, and only the Cas9-expressing and influenza-infected cells are affected.
Data Source
AI summary
Described herein are compositions and methods for a screening approach for identifying host factors that impact influenza viral production after the initial infection. Host factors that enhance influenza virus production were identified. Screening methods described herein include variations of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, termed CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi).