Polyvalent Guide RNAs for Broad-Spectrum CRISPR Antivirals
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Solution Overview
Problem
Current CRISPR antiviral systems face challenges in effectively targeting a broad range of viral strains due to rapid mutation rates and limited tolerance to polymorphisms, and existing methods for designing guide RNAs (gRNAs) do not adequately address the need for broad-spectrum antiviral activity while minimizing off-target effects in human genomes.
Innovation Solution
The development of polyvalent guide RNAs (pgRNAs) that exploit the promiscuity of CRISPR effectors to target multiple sites within a viral genome, using a single gRNA sequence designed to maximize activity across clinical strain variants while minimizing activity at human off-target sites, by calculating homology scores and relative activity scores to identify suitable target pairs and generate pgRNA templates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a single gRNA is designed to target multiple viral sites (polyvalent gRNA), then the breadth and efficiency of CRISPR antiviral activity is enhanced, but the complexity of gRNA design and selection increases
Solution Approach 1:
The patent applies universality by designing polyvalent gRNAs that can target multiple viral sites simultaneously. The gRNA sequence is engineered to recognize and bind to multiple different viral nucleic acid sequences through complementary base pairing, enabling a single gRNA to perform multiple antiviral functions against different viral targets or variants.
Solution Approach 2:
The patent employs parameter changes by systematically varying the gRNA sequence parameters (nucleotide composition, spacing, complementarity patterns) to optimize binding affinity and specificity across multiple target sites. The design process adjusts sequence parameters to maximize antiviral breadth while maintaining acceptable off-target profiles.
2Reliability
If multiple gRNAs are used to target different viral regions simultaneously, then mutational escape is suppressed, but the device complexity and difficulty of detecting and measuring off-target effects increase
Solution Approach 1:
The patent merges the functions of multiple gRNAs into a single polyvalent gRNA that simultaneously targets multiple viral regions. This consolidation maintains the ability to suppress mutational escape through multi-site targeting while simplifying the system to a single gRNA molecule, thereby reducing the complexity of detecting and measuring off-target effects compared to managing multiple separate gRNAs.
3Object-affected harmful factors
If CRISPR effectors are designed with high specificity to minimize off-target effects, then off-target activity is reduced, but the ability to tolerate polymorphisms in viral strains decreases
Solution Approach 1:
The patent applies local quality by designing gRNAs with position-specific complementarity patterns. Different regions of the gRNA have varying degrees of complementarity to the target sequence, with critical binding regions having high specificity and other regions having more flexible matching. This allows the gRNA to tolerate polymorphisms in certain positions while maintaining sufficient binding affinity and specificity at critical sites.
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 enhances the breadth and efficiency of CRISPR antiviral activity by allowing a single gRNA to target multiple viral sites, reducing the risk of mutational escape and off-target effects, thereby providing a more effective antiviral therapy and diagnostic tool.
Implementation Method 1
use a modular segment of their RNA cofactors known as CRISPR RNAs (crRNAs) or guide RNAs (gRNAs) to recognize and trigger the degradation of nucleic acids with a sequence complementary to that segment
Implementation Method 2
These diverse enzymes are derived from a bacterial and archaeal defensive response to invasive plasmids and viruses and, because of their ability to be easily redirected to nucleic acid with different sequences by simply changing the sequence composition of that short portion of their gRNAs called their 'spacer,' they have been re-appropriated over the past several years for a number of different biotechnological applications, most notably in precision gene editing. During precision gene editing, a CRISPR effector is transfected into a human cell and directed to introduce a double strand break (DSB) into the genomic DNA at a specific targeted sequence
Implementation Method 3
Cas13 nucleases also exhibit nonspecific RNAse activity after recognition of their targets, and this nonspecific degradation has been exploited in sensitive viral detection strategies as well
Data Source
AI summary
Generally, the present disclosure is directed to methods for gRNA design and products thereof that can be used as antivirals in which the produced gRNAs can be tolerant to polymorphisms across clinical strains and/or adapted for activity at multiple viral sites. Aspects of example gRNAs can also include reduced interactions with the human genome or transcriptome.