CRISPR gRNA Library Generation Using Non-Palindromic Restriction Sites
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Solution Overview
Problem
Current methods for generating CRISPR guide RNA (gRNA) libraries are costly, time-consuming, and prone to low yields and failures due to their complex nature, requiring multiple expensive kits and enzymes and involving extensive protocols.
Innovation Solution
The development of polynucleotides encoding for CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) with non-palindromic recognition sites for type II restriction enzymes, allowing for rapid and inexpensive generation of CRISPR gRNA libraries through simplified protocols that reduce the number of steps and time required.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional enzymatic methods are used to generate CRISPR gRNA libraries, then the libraries can be created with high specificity, but the process requires over 20 steps, multiple expensive kits and enzymes, and takes at least three days to complete
Solution Approach 1:
The invention divides the gRNA library generation process into modular components: a constant region polynucleotide with a specific non-palindromic recognition site, and variable spacer sequences. This segmentation allows for simplified assembly while maintaining the specificity required for accurate CRISPR guide RNA generation, reducing the overall protocol complexity from over 20 steps to a more manageable process.
Solution Approach 2:
The invention uses a standardized constant region polynucleotide template that can be repeatedly copied and combined with different variable spacer sequences. This copying approach eliminates the need to design and optimize unique complex protocols for each library generation, allowing high specificity to be maintained through template fidelity while dramatically reducing protocol complexity and time requirements.
2Manufacturing precision
If traditional enzymatic methods are used to generate CRISPR gRNA libraries, then the libraries can be created with high specificity, but the process requires extensive wash steps and takes at least three days to complete
Solution Approach 1:
The constant region polynucleotide is pre-designed with a specific non-palindromic recognition site for type II restriction enzymes, and the molecular weight tag is incorporated in advance. This preliminary preparation eliminates the need for multiple subsequent wash steps and extensive purification procedures, maintaining library generation specificity while reducing the overall process time from three days to a much shorter duration.
Solution Approach 2:
The invention changes the key parameter of the recognition site from palindromic to non-palindromic, which fundamentally alters the enzymatic processing requirements. This parameter change allows type II restriction enzymes to cut at predictable positions without requiring extensive wash steps, thereby maintaining specificity while dramatically reducing the time required for library generation.
3Adaptability or versatility
If traditional methods are used to generate CRISPR gRNA libraries, then comprehensive genome coverage can be achieved, but the cost is very high due to multiple expensive kits and enzymes
Solution Approach 1:
The constant region polynucleotide with the non-palindromic recognition site serves as a universal template that can be combined with any variable spacer sequence targeting any gene in the genome. This universal design maintains comprehensive genome coverage capability while eliminating the need for multiple expensive specialized kits and enzymes, as the same constant region and restriction enzyme system can be reused across all library generation projects.
Solution Approach 2:
The invention replaces expensive, complex enzymatic kits with a simpler system based on a reusable constant region polynucleotide template and standard type II restriction enzymes. The molecular weight tag enables easy identification without requiring expensive specialized reagents, making the overall process cost-effective while maintaining the ability to achieve comprehensive genome coverage through the variable spacer sequences.
4Manufacturing precision
If traditional enzymatic methods are used to generate CRISPR gRNA libraries, then the libraries can be created with high specificity, but the protocol involves over 20 steps with low yields and frequent failures
Solution Approach 1:
By segmenting the gRNA construct into a standardized constant region with built-in non-palindromic recognition site and separate variable spacer sequences, the invention eliminates many of the error-prone steps in traditional methods. This segmentation maintains the specificity required for accurate guide RNA generation while improving yield by reducing the number of manipulation steps where failures can occur.
Solution Approach 2:
Changing the recognition site parameter from palindromic to non-palindromic fundamentally simplifies the enzymatic processing, allowing type II restriction enzymes to cut at predictable positions without requiring multiple wash steps and extensive purification. This parameter change maintains library generation specificity while dramatically improving productivity by reducing low-yield steps and frequent failures associated with complex protocols.
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 the efficient and cost-effective creation of CRISPR gRNA libraries with increased yield and reduced complexity, allowing for the generation of complex libraries in a shorter timeframe, such as 3 hours, compared to traditional methods.
Implementation Method 1
a non-palindromic recognition site for a type II restriction enzyme/restriction endonuclease, the non-palindromic recognition site being oriented in a manner recognized by the type II restriction enzyme for cutting a site that is 17 to 27 base pairs past an end of the polynucleotide
Data Source
AI summary
The disclosure generally relates to compositions, polynucleotides, kits, methods, and systems for generating clustered regularly interspaced short palindromic repeats (CRISPR) libraries. Disclosed are polynucleotides encoding for a constant region of a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) having a non-palindromic recognition site for a type II restriction enzyme oriented in a manner recognized by the type II restriction enzyme for cutting a site past an end of the polynucleotide. The methods include using the polynucleotide to prepare CRISPR libraries.