De Novo gRNA Library Synthesis for Uniform Representation
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Solution Overview
Problem
Current methods for generating highly accurate and uniform nucleic acid libraries for targeted genome editing are inefficient, lacking in rapidity and precision, particularly in the design and optimization of custom pathways and organisms.
Innovation Solution
The development of systems and methods for de novo synthesis and screening of guide RNA (gRNA) libraries, comprising at least 500 non-identical DNA molecules, each encoding a different gRNA sequence, with a high percentage of molecules present within 2× of the mean frequency, and a GC base content of 20-85%, enabling precise editing of genes, gene clusters, or entire genomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to generate nucleic acid libraries, then the process can be completed, but the generation is inefficient and lacks rapidity
Solution Approach 1:
The patent employs template-directed synthesis where predetermined DNA templates containing multiple gRNA sequences are used as starting material. This preliminary preparation of templates allows for rapid amplification and generation of the final gRNA library without time-consuming sequential synthesis steps, thereby achieving high productivity with minimal time loss.
Solution Approach 2:
The invention uses PCR amplification to copy and replicate the gRNA sequences from the template DNA. This copying mechanism enables rapid generation of numerous identical gRNA molecules from a single template, dramatically increasing the generation speed and productivity of the nucleic acid library compared to conventional methods.
2Manufacturing precision
If conventional methods are used to generate nucleic acid libraries, then the process can be completed, but the accuracy and uniformity are insufficient
Solution Approach 1:
The patent designs specific DNA templates with predetermined gRNA sequences that are locally optimized for accurate transcription. The template structure includes specific features such as T7 promoter regions and designated gRNA coding sequences that ensure high-fidelity transcription. This local quality control at the template level propagates through to the final gRNA library, ensuring high accuracy and uniformity of sequence representation.
Solution Approach 2:
The invention incorporates quality control measures where the synthesized gRNA library is screened and analyzed to verify sequence accuracy and representation uniformity. Any deviations from the predetermined sequences can be detected and addressed, ensuring that the final library meets the required standards for reliability and consistency in gRNA representation.
3Quantity of substance
If large libraries with high diversity are generated, then the coverage of sequence space increases, but the uniformity of representation decreases
Solution Approach 1:
The patent prepares template DNA molecules that contain predetermined sets of gRNA sequences with controlled frequencies and representations. By pre-planning the template composition to include multiple copies of each gRNA sequence in a controlled manner, the method ensures that when amplification occurs, the final library maintains uniform representation across all sequence variants, thereby achieving both high diversity and compositional stability.
Data Source
AI summary
Disclosed herein are methods for the generation of nucleic acid libraries encoding for gRNA sequences. The gRNAs encoded by methods described herein may be single or double gRNA sequences. Methods described provide for the generation of gRNA libraries, as a DNA precursor or as a RNA transcription product, with improved accuracy and uniformity.