Random sgRNA Library Preparation via Restriction Enzyme Cleavage
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Solution Overview
Problem
Current methods for preparing sgRNAs for CRISPR applications are limited by low throughput, high cost, and the need for large-scale design and synthesis, particularly when targeting large genomic regions or entire genomes.
Innovation Solution
A method for preparing a random sgRNA full-coverage group of a target sequence involves cleaving DNA with a restriction enzyme, ligating an sgRNA skeleton, using a collateral activity restriction enzyme to acquire a protospacer DNA, phosphorylating the 5' end, ligating a T7 promoter, amplifying, and finally obtaining the sgRNA library through in vitro transcription.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional method (T7 promoter synthesis + overlap PCR + in vitro transcription) is used, then the process is simple and controllable, but the throughput is low and cannot handle large-scale sgRNA design
Solution Approach 1:
The patent segments the sgRNA preparation process into distinct modular steps: (1) DNA fragmentation and end repair, (2) sgRNA skeleton ligation, (3) protospacer extraction via collateral activity restriction enzyme, (4) T7 promoter ligation, (5) library amplification, and (6) in vitro transcription. This segmentation enables parallel processing and automated handling of large-scale sgRNA libraries without increasing overall process complexity
Solution Approach 2:
The patent introduces an intermediary sgRNA skeleton template that serves as a carrier for multiple protospacers. The skeleton contains collateral activity restriction enzyme sites that enable systematic extraction of protospacers through restriction enzyme digestion, facilitating high-throughput library construction without requiring individual sgRNA synthesis for each target
2Quantity of substance
If large-scale sgRNA design and synthesis is performed for whole genome coverage, then complete coverage is achieved, but the cost and technical challenge increase significantly
Solution Approach 1:
The patent creates a universal sgRNA skeleton template that can serve as a platform for generating multiple sgRNAs targeting different protospacers. The skeleton contains universal features (T7 promoter, collateral activity restriction enzyme sites) that enable systematic generation of thousands of sgRNAs through a single standardized workflow, eliminating the need for individual sgRNA design and synthesis for each target
Solution Approach 2:
The patent uses in vitro transcription to create multiple copies of sgRNAs from a single DNA template library. After amplifying the sgRNA skeleton library containing all protospacers, the system performs in vitro transcription to generate the final sgRNA pool, efficiently copying the required number of sgRNAs without requiring proportional physical synthesis of each individual sgRNA
3Manufacturing precision
If restriction enzyme is used to identify PAM sequence and flatten end, then uniform coverage is achieved, but additional enzymatic steps are required
Solution Approach 1:
The patent merges multiple functions into the restriction enzyme step: (1) identification and cleavage at PAM sequences, (2) generation of blunt ends through flattening, and (3) creation of uniform fragmentation patterns. By combining these functions into a single enzymatic action using restriction enzymes like SerFI, MspI, HpaII, BstNI, BfaI, or DdeI, the system achieves uniform coverage without requiring separate steps for each function
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 enables the preparation of a random sgRNA library that provides uniform coverage of a target sequence with low preference, independent of target sequence length, and without the need for large-scale sgRNA design, thus overcoming the limitations of existing methods.
Implementation Method 1
cleaving a sample DNA using a restriction enzyme for identifying a PAM sequence and flattening an end
Implementation Method 2
the end is flattened using Mung Bean Nuclease
Implementation Method 3
ligating an sgRNA skeleton to a flattened 3' end of the double-stranded DNA obtained in step (1)
Implementation Method 4
cleaving a ligation product in step (2) using the collateral activity restriction enzyme, acquiring a protospacer DNA
Implementation Method 5
ligating a sequence of a T7 promoter to the 5' end of the protospacer DNA
Implementation Method 6
obtaining an sgRNA library through in vitro transcription
Data Source
AI summary
Provided is a method for preparing a random sgRNA full-coverage group of a target sequence. The method comprises: cleaving a PAM region of a target sequence using a restriction enzyme; linking same to an sgRNA skeleton; acquiring a sequence targeting a protospacer region by means of a restriction enzyme site having a collateral activity on the sgRNA skeleton; linking same to a T7 promoter; acquiring an sgRNA library with the T7 promoter by means of amplification; and acquiring an sgRNA library of the target sequence by means of in-vitro transcription. Also disclosed are a method for preparing an sgRNA library of ribosomal RNA, a method for removing ribosomal RNA from the RNA library, and a method for removing human whole genomes from a host genome. The method has the advantages of low cost, simple manufacture, uniform coverage, low preference, no limitation on the length of a target sequence, no need for design of sgRNAs in large quantities, etc.