CRISPR Nickase Labeling for Genome Assembly
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Solution Overview
Problem
Current genome analysis methods, such as de novo genome sequence assembly and structural variation analysis, face challenges in achieving long-range contiguity, accurately mapping repetitive regions, and detecting structural variants due to limitations in sequencing technologies and the lack of appropriate sequence motifs in repetitive genomic areas.
Innovation Solution
The use of CRISPR/Cas9 nickase technology for sequence-specific labeling, which involves guiding a Cas9 nickase with a guide RNA to introduce single-strand breaks in specific genomic locations, followed by incorporation of fluorescently labeled nucleotides to target and map repetitive regions and structural variations, enabling precise labeling and mapping of repetitive sequences and structural variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing approaches (mate-pair libraries, fosmid/BAC clone libraries) are used to improve sequence contiguity, then assembly quality improves, but device complexity and labor requirements increase significantly
Solution Approach 1:
The patent introduces CRISPR/Cas9 as an intermediary system that uses guide RNA to direct specific DNA cleavage at target sequences. This mediator approach replaces complex library preparation methods with a simpler, sequence-specific nicking strategy that achieves long-range mapping without requiring fosmid or BAC clone libraries
Solution Approach 2:
The patent changes the fundamental parameter of DNA cleavage from random or motif-dependent (conventional restriction enzymes) to sequence-specific targeting via CRISPR guide RNA. This parameter change enables direct targeting of repetitive regions and structural variants without relying on complex library construction
2Ease of operation
If optical mapping with restriction enzymes is used to provide scaffolds for genome assembly, then scaffolding capability improves, but measurement precision deteriorates in repetitive regions due to lack of sequence motifs
Solution Approach 1:
The patent uses guide RNA as an intermediary that bridges the gap between CRISPR/Cas9 and target DNA sequences in repetitive regions. The guide RNA provides sequence-specific recognition where conventional restriction enzymes fail due to lack of motifs, enabling precise mapping in previously inaccessible regions
Solution Approach 2:
The patent extracts the sequence-specific recognition function from conventional restriction enzymes and transfers it to the CRISPR/Cas9-guide RNA system. This extraction allows targeting of any sequence defined by the guide RNA, including repetitive regions that lack restriction sites
3Measurement precision
If deep sequencing of shotgun libraries is performed to improve assembly quality, then sequence contiguity improves, but loss of time and productivity increase
Solution Approach 1:
The patent performs preliminary action by using CRISPR/Cas9 to introduce nicks at specific target sequences before sequencing. This pre-marking of genomic locations with fluorescent labels allows direct detection of structural variants and simplifies assembly, reducing the need for extensive deep sequencing
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 allows for accurate and efficient detection and mapping of repetitive sequences and structural variants, improving genome assembly and structural variation analysis by providing high-resolution, sequence-specific information, especially in regions previously inaccessible due to lack of sequence motifs.
Implementation Method 1
contacting genomic DNA with a guide RNA having a portion complementary to the target sequence in the genomic DNA
Implementation Method 2
with Cas9 nicking endonuclease (nickase) to produce a single-strand break (nick) in the genomic DNA at a specific location adjacent to the target sequence
Implementation Method 3
contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide. The fluorescently labeled nucleotide is incorporated into the nicked DNA at the specific location
Implementation Method 4
The target nucleic acid sequence is detected via fluorescent label
Data Source
AI summary
Provided herein are methods of detecting a target nucleic acid sequence. In one embodiment, the method includes contacting genomic DNA with a guide RNA having a portion complementary to the target sequence in the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at a specific location adjacent to the target sequence. The method further includes contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide. The fluorescently labeled nucleotide is incorporated into the nicked DNA at the specific location and the target nucleic acid sequence is detected via fluorescent label.