CRISPR Guide RNA Library Generation via Endonuclease Cleavage
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Solution Overview
Problem
There is a need for cost-effective methods and compositions to generate sets of guide RNAs that can target specific loci within a given target nucleic acid, particularly for generating libraries of CRISPR/Cas guide RNAs for genome editing and labeling applications.
Innovation Solution
The development of methods and compositions for generating a library of DNA molecules encoding CRISPR/Cas guide RNAs, including Cas9 single guide RNAs or Cas9 targeter RNAs, using DNA endonucleases to cleave target DNA and attach adapters, followed by attachment of a constant region to generate DNA molecules encoding CRISPR/Cas guide RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used to generate guide RNA libraries, then guide RNAs can be produced, but the process is costly and time-consuming
Solution Approach 1:
The patent performs preliminary actions by pre-designing and synthesizing DNA adapters with specific overhang sequences that correspond to CRISPR spacers. These adapters are prepared in advance with restriction enzyme sites and homology regions, allowing rapid cloning of guide RNA sequences without time-consuming de novo synthesis for each guide RNA variant.
Solution Approach 2:
The patent uses copying by synthesizing DNA adapters that contain copies of CRISPR spacer sequences from the target genome. These adapter copies are then cloned into expression vectors using restriction enzyme digestion and ligation, enabling rapid generation of multiple guide RNA variants through replication of the adapter-template structure rather than individual synthesis.
2Adaptability or versatility
If comprehensive guide RNA libraries are generated to cover entire genomes, then all possible target loci can be addressed, but the complexity and cost of the process increases significantly
Solution Approach 1:
The patent segments the genome into discrete CRISPR spacer sequences that can be individually adapted to guide RNA expression vectors. By dividing the comprehensive genome coverage into modular spacer-adapter units, the system achieves complete locus coverage while maintaining manageable complexity through standardized adapter designs with universal restriction sites and homology regions.
Solution Approach 2:
The patent applies universality through DNA adapters that serve multiple functions: they contain CRISPR spacer sequences for target recognition, restriction enzyme sites for cloning, and homology regions for efficient ligation. This multi-functional adapter design allows the same basic structure to be used across the entire genome, simplifying the library generation process while maintaining comprehensive coverage.
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 the efficient generation of CRISPR/Cas guide RNA libraries, allowing for precise targeting and modification of nucleic acids, facilitating genome editing and labeling applications by providing a versatile tool for site-specific activity and locus-specific regulation.
Implementation Method 1
contacting target DNA with one or more DNA endonucleases that specifically bind to and cleave within a recognition sequence
Implementation Method 2
a DNA ligase, to join the adapter to the cleavage fragments
Data Source
Figure 1A~1B
Figure 1C~1E
Figure 2A~2B
AI summary
The present disclosure provides methods, kits, and compositions for generating DNA molecules encoding CRISPR/Cas guide RNAs (e.g., Cas9 single guide RNAs or Cas9 targeter RNAs). A library of such DNA molecules can be generated from any DNA source. The methods include a step of contacting target DNA with one or more DNA endonucleases that specifically bind to and cleave within a recognition sequence that includes a PAM sequence, to generate a plurality of cleavage fragments, to which a DNA adapter can be attached. A distal-cleaving DNA endonuclease can be used that specifically binds to a recognition sequence in the DNA adapter and cleaves at a site within the attached DNA cleavage fragments to generate a library of CRISPR/Cas guide sequences. After removal of all or a portion of the DNA adapter, a constant region of a guide RNA can be attached to generate DNA molecules encoding CRISPR/Cas guide RNAs.