CRISPR-Cas Enzyme Engineering Framework
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Solution Overview
Problem
Current methods for enzyme engineering face challenges in efficiently screening and creating libraries of enzyme variants with high activity, due to limitations in identifying suitable mutation residues and the complexity of screening processes.
Innovation Solution
The use of CRISPR-Cas technology to direct the formation of CRISPR complexes in bacterial cells, enabling the introduction of specific and random mutations into genes of interest using engineered pCas9 plasmids, which include dCas9 enzymes with deaminase activity and Cas9 enzymes, allowing for precise and efficient mutation induction without extracting the gene from the plasmid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional enzyme engineering methods are used to screen and create enzyme variant libraries, then mutations can be introduced into enzymes, but the process is time-consuming and limited in the range of mutations that can be efficiently screened
Solution Approach 1:
The patent replaces traditional mechanical/screening-based enzyme engineering methods with CRISPR-Cas9 genome editing technology. Instead of relying on random mutagenesis and laborious screening, the invention uses programmable guide RNAs to direct Cas9 nuclease to specific genomic locations, enabling precise and rapid introduction of mutations directly in vivo, thereby dramatically accelerating the creation of enzyme variant libraries.
Solution Approach 2:
The invention changes the fundamental parameter of mutation introduction from random/low-frequency natural mutation to targeted/high-frequency CRISPR-mediated mutation. By modifying the mutation rate and specificity parameters through guide RNA design, the system can rapidly generate diverse enzyme variants with desired properties, overcoming the time limitation of traditional screening approaches.
2Manufacturing precision
If CRISPR-Cas9 is used to introduce multiple specific mutations into a gene, then precision of mutation placement is improved, but the complexity of the editing system increases
Solution Approach 1:
The patent divides the complex task of introducing multiple mutations into separate, manageable components: individual guide RNAs each targeting specific genomic locations. Each guide RNA-Cas9 complex operates independently to introduce a specific mutation, allowing precise control over mutation placement while simplifying the overall design and implementation of the editing system.
Data Source
AI summary
The invention describes a method for utilizing the CRISPR-Cas system to edit any gene of interest present on a plasmid. The method uses CRISPR tool to engineer enzymes for better activity by allowing a cell to undergo specific and random mutations. Described methods include newly designed, engineered and modified vector systems, which encodes single or multiplex gene targets and Cas9/deaminase Cas9 proteins. The invention is useful for single or multiple gene editing for industrial applications such as to edit genes encoding antibiotics, therapeutic proteins or any important industrial enzymes. The invention is a quick and efficient tool for creating enzyme variant libraries containing a vast range of permutation and combination of mutation that will be assayed for highest activity. Hot spots will be identified on gene of interest which will aid in generating mutations in these places. These mutations can be rationalized in specific places or random single base substitutions.