CRISPR Multiplexing via Shared Target Sequences
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Solution Overview
Problem
Current CRISPR/Cas systems face limitations in efficiently targeting and editing multiple loci within a genome, particularly when dealing with essential genes, as they often require separate guide RNAs and can introduce scars or leave behind residual Cas9 activity.
Innovation Solution
Genetically modified cells and organisms with integrated heterologous CRISPR/Cas target sequences at multiple positions within their genome, allowing a single guide RNA to target multiple sites, enabling simultaneous gene editing and optional removal of the Cas9 expression cassette for scarless integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If separate guide RNAs are used to target multiple loci, then each locus can be specifically targeted, but the complexity of the system increases and efficiency decreases
Solution Approach 1:
The patent applies universality by designing a single guide RNA that can target multiple loci simultaneously. The guide RNA contains a spacer sequence complementary to a shared target sequence that is integrated at multiple genomic positions, allowing one guide RNA to direct Cas9 to multiple different locations in the genome, thereby reducing system complexity while maintaining multiplexing capability
Solution Approach 2:
The patent applies segmentation by dividing the genome into multiple locations that share a common target sequence. Instead of requiring separate guide RNAs for each locus, the genome is segmented such that multiple loci contain the same or highly similar target sequence, enabling a single guide RNA to address multiple segments simultaneously
2Ease of operation
If Cas9 expression cassette is retained for continuous editing, then editing can be performed on demand, but residual Cas9 activity causes off-target effects and toxicity
Solution Approach 1:
The patent applies periodic action by implementing a two-stage process: first, Cas9 is expressed to perform the desired genome editing; second, the Cas9 expression cassette is removed or inactivated. This periodic expression pattern allows the system to have Cas9 activity only when needed, eliminating residual activity that causes off-target effects while maintaining on-demand editing capability during the active phase
Solution Approach 2:
The patent applies preliminary action by designing the system so that the Cas9 expression cassette is removed or inactivated after the editing is complete. This preliminary removal step is built into the system design, ensuring that Cas9 activity is terminated before it can cause harmful off-target effects, while the editing function was already performed during the active period
3Manufacturing precision
If multiple guide RNAs are used to target multiple loci, then each locus receives precise targeting, but the number of components increases and integration becomes scarred
Solution Approach 1:
The patent applies universality by creating a single guide RNA that performs multiple targeting functions. The guide RNA is designed with a spacer sequence that matches a shared target sequence present at multiple loci, allowing one guide RNA component to precisely target multiple different locations simultaneously, thereby reducing the number of components while maintaining targeting precision
Solution Approach 2:
The patent applies merging by combining multiple target sequences into a single shared target sequence that is integrated at multiple genomic positions. Instead of using separate guide RNAs for each locus, the system merges the targeting function into a single guide RNA that recognizes the shared sequence, reducing component number while achieving multiplexed precise editing
4Productivity
If the same target sequence is integrated at multiple positions, then a single guide RNA can target multiple sites, but the integration process becomes more complex
Solution Approach 1:
The patent applies preliminary action by pre-integrating the shared target sequence at multiple genomic positions before the CRISPR editing step. This preliminary integration creates a standardized target that can be recognized by a single guide RNA, simplifying the subsequent editing process and enabling efficient multiplexing without requiring complex real-time targeting strategies
Solution Approach 2:
The patent applies parameter changes by modifying the genome to include identical or highly similar target sequences at multiple locations. This parameter change (making target sequences uniform across multiple loci) enables a single guide RNA to target multiple sites simultaneously, improving multiplexing efficiency despite the initial complexity of the integration process
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 precise, efficient, and scarless multiplexing of gene editing at multiple loci, including essential genes, with reduced toxicity and improved genome editing efficiency using a single guide RNA, while allowing for the removal of Cas9 to prevent off-target effects.
Implementation Method 1
A guide RNA includes (i) a nucleotide sequence (a guide sequence) that is complementary to a sequence (the target site) of a target DNA
Implementation Method 2
Wild type CRISPR/Cas proteins (e.g., a Cas9 protein) normally have nuclease activity that cleaves a target nucleic acid (e.g., a double stranded DNA (dsDNA)) at a target site
Implementation Method 3
A DSB can be repaired via homologous recombination (often from amplified polymerase chain reaction (PCR) fragments or vectors) to create a modification (deletion, addition, allele insertion, etc.) with high precision and efficiency
Data Source
AI summary
The present disclosure provides genetically modified cells and non-human organisms, and methods of producing such cells and organisms. Also provided are methods of editing the genome of such cells and organisms. The cells and non-human organisms of the disclosure are genetically modified such that their genome includes an integrated heterologous nucleic acid (that includes a CRISPR/Cas target sequence) at one or more (e.g., 2 or more, 3 or more, 4 or more, etc.) positions within the genome. The integrated nucleic acids, which include the same CRISPR/Cas target sequence, allow for simultaneous gene editing at multiple different positions within a genome using a single species of CRISPR/Cas guide RNA (i.e., one guide RNA will target multiple sites because the multiple sites have the same target sequence).