CRISPR gRNA Design for Variant Gene Knockout
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Solution Overview
Problem
There is a need to improve the efficiency of producing functional knockouts in gene editing using CRISPR/Cas systems, particularly in reducing the expression or activity of variant genes in cancer cells.
Innovation Solution
The method involves introducing guide RNAs (gRNAs) and CRISPR-associated endonucleases into cells to target specific sequences in variant genes, including those with protospacer adjacent motifs (PAM) sites created by mutations, and intronic PAM sites, to reduce gene expression or activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional CRISPR/Cas systems are used to target wild-type genes, then gene editing capability is achieved, but efficiency of producing functional knockouts is insufficient
Solution Approach 1:
The patent applies local quality by designing gRNAs that specifically target mutant sequences rather than wild-type sequences. The first gRNA targets a mutant-specific sequence created by the mutation, while the second gRNA targets a nearby sequence, creating locally optimized targeting that exploits the specific genetic alteration in cancer cells to achieve efficient knockout.
Solution Approach 2:
The patent segments the knockout strategy into two separate gRNAs: one targeting the mutant-specific sequence and another targeting a nearby sequence. This segmentation allows independent optimization of each target site and increases overall knockout efficiency by creating multiple cleavage sites that work synergistically.
2Adaptability or versatility
If gRNAs target mutated sequences in variant genes, then specificity to cancer cells is improved, but complexity of designing effective gRNA pairs increases
Solution Approach 1:
The patent applies preliminary action by pre-identifying mutant-specific sequences that contain or create PAM sites near the mutation. This preliminary identification simplifies subsequent gRNA design by providing ready-made target sequences that are guaranteed to be specific to the mutant allele, reducing the complexity of finding suitable target pairs.
Solution Approach 2:
The patent uses the mutant sequence itself as an intermediary element that facilitates gRNA design. The mutation creates or exposes a PAM site or nearby sequence that serves as a natural anchor point for designing the first gRNA, making the design process more systematic and less complex.
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 effectively reduces the expression or activity of variant genes in cells, including cancer cells, by inducing targeted cleavage and exon skipping, thereby potentially treating cancer by disabling cancer-driving genes.
Implementation Method 1
one or more of the gRNAs hybridizes to a target sequence comprising a protospacer adjacent motif (PAM) site in the variant gene
Implementation Method 2
the one or more CRISPR-associated endonucleases cleave the variant gene at the target sequences
Data Source
AI summary
This disclosure relates to methods of and compositions for reducing expression or activity of a variant gene comprising at least one mutation as compared its wild-type gene, comprising introducing into a cell comprising the variant gene one or more DNA sequences encoding two or more gRNAs that are complementary to two or more target sequences in the variant gene, wherein at least one of the gRNAs hybridizes to a target sequence comprising a PAM site in the variant gene that results from a mutation to the variant gene creating the PAM site that does not exist in the wild-type gene or is operably linked to a mutated portion of the wild-type gene, at least one of the gRNAs hybridizes to a target sequence comprising a PAM site in an intron of the variant gene downstream or upstream from the PAM site, and a nucleic acid sequence encoding a CRISPR-associated endonuclease; wherein a CRISPR-associated endonuclease cleaves the variant gene at the target sequences; and expression or activity of the variant gene is reduced in the cell relative to a cell in which the one or more DNA sequences encoding the two or more gRNAs and the nucleic acid sequence encoding the CRISPR-associated endonuclease are not introduced.