Engineered Cas9 Protein for Precise Single-Base Editing
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Solution Overview
Problem
Current CRISPR-Cas9 systems face challenges in achieving precise genomic modifications, particularly in requiring diverse tools for recognizing and targeting various genomic sequences, and struggle with making single-base edits within genes.
Innovation Solution
Development of an engineered, non-naturally occurring Cas9 protein from Lachnospira bacteria with specificity for unique protospacer adjacent motifs (PAM) sequences, such as 5′-NNGNG-3′, which can be expressed in eukaryotic cells and used to target specific genomic sites with high sequence identity and mutations for enhanced precision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR-Cas9 systems are used to knock out or modify gene expression, then genetic engineering capabilities are improved, but precise single-base editing within genes remains challenging
Solution Approach 1:
The patent applies parameter changes by modifying the PAM sequence recognition specificity from the conventional NGG to NAG. This parameter change in the recognition sequence allows the Cas9 variant to target different genomic locations, enabling precise single-base editing while maintaining the ability to perform broader gene editing operations. The altered PAM specificity expands the reachable target space while allowing for high-precision modifications.
2Adaptability or versatility
If diverse CRISPR tools are used to target various genomic sequences, then the CRISPR toolbox is expanded, but the complexity of requiring multiple different tools increases
Solution Approach 1:
The patent applies universality by engineering a single Cas9 variant with altered PAM specificity that can serve multiple functions. This single modified Cas9 protein can target both traditional NGG sites and the new NAG sites, effectively performing the role of multiple different Cas proteins. The engineered Cas9 maintains compatibility with existing guide RNA designs while adding new targeting capabilities, reducing the need to maintain separate toolkits for different PAM specificities.
3Adaptability or versatility
If engineered Cas9 proteins with new PAM specificities are developed, then the range of targetable genomic sites is improved, but the difficulty of engineering and expressing functional proteins increases
Solution Approach 1:
The patent applies parameter changes by making specific amino acid substitutions in the Cas9 protein structure that alter PAM recognition from NGG to NAG. These targeted parameter changes in the protein sequence enable the engineered Cas9 to recognize new genomic sites while maintaining proper folding and function in eukaryotic cells. The modifications are designed to be minimal and focused, facilitating easier engineering compared to creating entirely new Cas proteins.
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
Enables precise gene editing and modification by allowing the Cas9 protein to recognize and bind to specific PAM sequences, facilitating efficient cleavage or editing of target nucleic acids in eukaryotic cells, including human cells, with high specificity and accuracy.
Implementation Method 1
the Cas9 protein recognizes a specific PAM sequence defined by 5'-NNGNG-3'
Implementation Method 2
CRISPR-Cas9 can be used to knock out a gene or modify the expression of a gene
Data Source
AI summary
The present invention provides novel systems, methods and compositions for making and using a recombinantly engineered novel Cas9 optimized for human cells, for nucleic acid targeting and manipulation. The present invention is based on the discovery of a novel Cas9 species from Lachnospira bacterium that was codon-optimized and recombinantly produced for use in human ceils. In some embodiments, the novel Cas9 can be used in a base editor. In some embodiments, the novel engineered Cas9 is used to treat human diseases.