Enzymatic CRISPR Guide Strand Library Construction
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Solution Overview
Problem
Current methods for synthesizing customized CRISPR/Cas9 guide strand libraries are costly and time-consuming, requiring precise genomic information and taking several weeks to produce, necessitating a more efficient and cost-effective approach.
Innovation Solution
The development of a method involving polynucleotides that encode CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) with non-palindromic recognition sites for type II restriction enzymes, allowing for enzymatic construction of guide strand libraries through digestion and ligation steps combined in single reactions, and utilizing methyltransferase activity to prevent further cleavage, thereby reducing production time while maintaining accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If chemical synthesis methods are used to create customized CRISPR guide strand libraries, then library accuracy and customization are maintained, but production time increases to several weeks and costs become prohibitive
Solution Approach 1:
The patent replaces chemical synthesis methods with enzymatic construction using type II restriction enzymes with methylase activity. The enzymatic system uses methylated nucleotides to block further cleavage, enabling automated, high-precision library generation that maintains accuracy while reducing production time from weeks to days.
Solution Approach 2:
The patent changes the fundamental parameter of library construction from chemical synthesis to enzymatic digestion and ligation. By using type II restriction enzymes with methylase activity, the system achieves both high precision through specific recognition sites and rapid production through enzymatic catalysis, resolving the contradiction between accuracy and time.
2Manufacturing precision
If chemical synthesis methods are used to create customized CRISPR guide strand libraries, then customization precision is maintained, but costs become prohibitive
Solution Approach 1:
The patent replaces expensive chemical synthesis with cost-effective enzymatic construction. Type II restriction enzymes with methylase activity provide precise customization at lower costs by using natural enzymatic recognition and methylation mechanisms, making customized library production economically viable.
Solution Approach 2:
The enzymatic system performs self-service through autonomous recognition of methylated nucleotides and automatic blocking of cleavage sites. This self-regulating mechanism eliminates the need for expensive manual intervention and quality control steps required in chemical synthesis, reducing overall production costs while maintaining precision.
3Adaptability or versatility
If traditional library construction methods are used, then comprehensive genomic information can be utilized, but the process requires precise genomic information on gene structure and nucleotide polymorphisms
Solution Approach 1:
The patent changes the information requirement parameter from needing precise genomic information to using general genomic sequences. The type II restriction enzyme with methylase activity automatically identifies and processes target sites through methylation, simplifying the process while maintaining the ability to utilize comprehensive genomic information for library construction.
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 method significantly reduces the cost and time required for library production while maintaining high fidelity and efficiency, enabling rapid generation of CRISPR/Cas guide strand libraries.
Implementation Method 1
The type II restriction enzyme has methylase activity and cleavage activity and the recognition site comprises a nucleotide which is methylated by said type II restriction enzyme upon cleavage, methylation of said nucleotide altering said recognition site such that the type II enzyme no longer binds said site
Implementation Method 2
a non-palindromic recognition site recognized by a type II restriction enzyme oriented in said sequence such that a second operably linked polynucleotide is cleaved 17 to 27 base pairs from said recognition site
Data Source
AI summary
A method for the rapid and efficient production of guide strand libraries such as CRISPR gRNA libraries is disclosed.