Combinatorial Cas9 Variant Screening Without Junction Sequence Scars
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Solution Overview
Problem
Current systems for generating and screening large numbers of genetic variants of recombinant proteins, such as Cas9, are cumbersome and inefficient, lacking high-throughput capabilities.
Innovation Solution
A high-throughput combinatorial genetic modification system, CombinSEAL, that seamlessly connects genetic components without introducing artificial sequences, allowing for the generation and screening of combinatorial mutants, including optimized Cas9 variants with improved on-target cleavage and reduced off-target activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional systems are used for generating and screening genetic variants, then the process can be completed, but the system is cumbersome, labor-intensive, and inefficient
Solution Approach 1:
The patent divides the genetic modification process into modular components: (1) a DNA template with multiple recognition sites for type IIS restriction enzymes, (2) barcode sequences for tracking variants, and (3) a systematic method for generating combinatorial mutants. This segmentation enables high-throughput generation of genetic variants by allowing parallel processing of multiple DNA constructs, directly addressing the throughput limitation of traditional systems.
Solution Approach 2:
The patent prepares DNA templates with pre-designed recognition sites for type IIS restriction enzymes before the actual variant generation process. The templates are engineered in advance with multiple cloning sites and barcode regions, allowing rapid assembly of combinatorial mutant libraries without time-consuming manual manipulation during the screening phase, thus improving overall efficiency.
2Adaptability or versatility
If combinatorial mutant libraries are generated, then diverse protein variants are obtained, but artificial or extraneous amino acid sequences are introduced at junction sites
Solution Approach 1:
The patent applies local quality control by designing type IIS restriction enzyme recognition sites positioned such that the enzyme cuts outside the coding sequence. This ensures that while combinatorial assembly occurs at junction sites, the actual amino acid coding regions maintain their native sequences without introducing extraneous residues, except for the intended combinatorial mutations.
Solution Approach 2:
The patent uses type IIS restriction enzymes as intermediaries to facilitate the assembly of DNA fragments. These enzymes recognize specific sequences and cut outside those sequences, generating compatible ends for ligation without leaving residual recognition sites or extraneous amino acid sequences in the final protein product, thus maintaining sequence purity while enabling combinatorial diversity.
3Productivity
If high-throughput screening is implemented, then efficient identification of optimized variants is achieved, but the system requires sophisticated methodology and resources
Solution Approach 1:
The patent uses barcode sequences as copies or tags that uniquely identify each genetic variant in the combinatorial library. These barcodes are sequenced in parallel to track and identify variants of interest, enabling high-throughput screening without requiring individual analysis of each mutant. This copying approach simplifies the screening system by replacing complex individual characterization with parallel barcode sequencing.
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 efficient generation and identification of recombinant proteins with enhanced functionalities, specifically SpCas9 mutants showing improved on-target cleavage and reduced off-target effects, facilitating rapid screening and optimization.
Implementation Method 1
a first recognition site for a first type IIS restriction enzyme; a first and a second recognition sites for a second type IIS restriction enzyme
Data Source
AI summary
The present invention provides to an improved high-throughput system and method for generated and screening of genetic variants by combinatorial modifications. Also provided are optimized SpCas9 enzyme variants produced by this system.