Multiplex CRISPR Array Assembly via Bridge Oligonucleotides
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Solution Overview
Problem
The technical challenge lies in efficiently assembling multiplex CRISPR arrays, particularly with natural CRISPR-Cas systems like Cas12a, due to the complexity and length of synthetic targeting arrays, which are difficult to synthesize and assemble, especially when using repetitive sequences that complicate commercial synthesis and laboratory assembly.
Innovation Solution
A method involving the use of bridge oligonucleotides to facilitate the hybridization and ligation of single-stranded DNA oligonucleotides, allowing for the rapid assembly of multiplex CRISPR arrays without the need for extensive sequence modifications or time-consuming cloning steps, by positioning oligonucleotides for correct ligation within spacer regions while avoiding incorrect annealing within repetitive sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If synthetic targeting arrays are used for multiplexing with CRISPR-Cas9 and its homologs, then multiple guide RNAs can be used in the same cell, but the arrays become very long and assembly becomes time-consuming
Solution Approach 1:
The patent divides the long synthetic targeting array into multiple shorter oligonucleotide fragments. Each oligonucleotide contains a portion of the CRISPR array sequence, and these fragments are assembled together through hybridization and ligation. This segmentation reduces the time required to synthesize and assemble the complete array while maintaining the ability to use multiple guide RNAs in the same cell.
2Length of stationary object
If natural CRISPR arrays with repetitive sequences are used, then the arrays become more compact, but they become difficult to synthesize commercially or assemble in the lab
Solution Approach 1:
The patent segments the compact natural CRISPR array into multiple shorter oligonucleotides, each containing a portion of the repetitive sequence. This segmentation makes synthesis commercially feasible while preserving the compact structure. The oligonucleotides are then assembled using bridge oligonucleotides that facilitate correct positioning without requiring synthesis of the entire repetitive array as a single unit.
Solution Approach 2:
The patent introduces bridge oligonucleotides as intermediaries to facilitate the assembly of oligonucleotides containing repetitive sequences. These bridge oligonucleotides hybridize to complementary sequences on adjacent oligonucleotides, positioning them correctly for ligation. This intermediary approach enables accurate assembly of compact arrays with repetitive sequences that would otherwise be difficult to synthesize or assemble.
3Productivity
If bridge oligonucleotides are used to facilitate hybridization and ligation, then assembly becomes rapid and accurate, but the process requires multiple oligonucleotide components
Solution Approach 1:
The patent uses bridge oligonucleotides as intermediary components that facilitate the hybridization and ligation of array oligonucleotides. Each bridge oligonucleotide contains sequences complementary to adjacent array segments, enabling them to anneal in the correct orientation. This intermediary approach accelerates assembly by providing predefined hybridization sites, reducing the need for extensive optimization of direct oligonucleotide interactions.
Solution Approach 2:
The patent segments the CRISPR array assembly process into distinct oligonucleotide components that can be independently synthesized and then assembled. This segmentation allows for standardized production of array fragments and bridge oligonucleotides, which can be combined in a modular fashion to create various CRISPR arrays, thereby improving productivity despite the increased number of components.
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 rapid, accurate, and cost-effective assembly of multiplex CRISPR arrays, improving their efficacy in genome editing and gene regulation applications by ensuring correct spacer order and array length, even for arrays with multiple spacers, and is compatible with various CRISPR systems, including Cas12a.
Implementation Method 1
allowing the first oligonucleotide and the second oligonucleotide to hybridize with the bridge oligonucleotide
Implementation Method 2
ligating the first and second oligonucleotide
Data Source
AI summary
Provided herein are methods for generating multiplex CRISPR arrays based on annealing and ligating single-stranded DNA oligonucleotides using bridge oligonucleotides. The methods described herein include providing a first oligonucleotide comprising a CRISPR repeat sequence or a portion thereof, and a first portion of a first spacer sequence at its 3′ end; providing a second oligonucleotide comprising, from 5′ to 3′, a second portion of the first spacer sequence, the CRISPR repeat sequence, and a first portion of a second spacer sequence; providing a bridge oligonucleotide comprising a sequence substantially complementary to the first spacer sequence; allowing the first oligonucleotide and the second oligonucleotide to hybridize with the bridge oligonucleotide; and ligating the first and second oligonucleotide.