CRISPR-nickase prokaryotic genome editing
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Solution Overview
Problem
Current methods for remodeling bacterial genomes lack efficient techniques for targeted large-scale genome editing, relying on recombinases or meganucleases that require exogenous sequences and are limited to single-gene knockouts, making it difficult to induce programmable and controllable recombination between endogenous DNA sequences.
Innovation Solution
The use of a CRISPR-Cas system with a nicking nuclease that introduces single-stranded breaks in the prokaryotic genome, allowing for targeted deletion or replacement of genomic regions by homologous recombination, using guide crRNAs complementary to regions near repeated sequences and protospacer adjacent motifs (PAMs), enabling the removal or replacement of sequences up to several kilobases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If recombinases or meganucleases are used for genome remodeling, then targeted deletion can be achieved, but exogenous sequences must be inserted into the bacterial genome and the method is limited to single-gene knockouts
Solution Approach 1:
The patent extracts and eliminates the requirement for exogenous recombinase or meganuclease sequences from the bacterial genome. By using CRISPR-nickase system with endogenous repeat sequences as targets, the method achieves targeted deletion without inserting external sequences, thereby simplifying the genetic architecture while maintaining precision.
Solution Approach 2:
The CRISPR-nickase system provides multi-functionality by enabling both single-gene knockouts and large-scale genome remodeling through a single platform. The system can target various endogenous repeat sequences (IS elements, transposons, etc.) to achieve different deletion scales without requiring different enzymatic tools, thus resolving the limitation of previous methods.
2Volume of moving object
If spontaneous stochastic DNA breaks are used for genome rearrangement, then large chromosomal rearrangements can occur, but the process is time-consuming and laborious due to lack of targeting capability
Solution Approach 1:
The patent applies preliminary action by pre-designing and introducing crRNA guide sequences that specifically target repeat sequences before the deletion event occurs. This programmed targeting eliminates the need to wait for spontaneous breaks, significantly reducing experiment time while enabling large chromosomal rearrangements through directed homology recombination.
Solution Approach 2:
The system incorporates feedback through the CRISPR-Cas mechanism where crRNA guides the nickase to specific target sequences, and the outcome is feedback through successful deletion verified by PCR or sequencing. This controlled feedback loop replaces random waiting with directed progression, accelerating large-scale rearrangement experiments.
3Adaptability or versatility
If CRISPR-Cas system with nicking nuclease is used, then targeted large-scale genome editing can be achieved without exogenous sequences, but the method requires precise targeting near repeated sequences and PAM motifs
Solution Approach 1:
The patent applies local quality by exploiting the specific properties of repeat sequences and PAM motifs at targeted locations. These endogenous elements provide unique recognition sites that enable precise targeting without requiring external sequences. The method leverages the natural distribution and characteristics of these sequences to achieve accurate, programmable edits at desired genomic locations.
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 precise and efficient editing of prokaryotic genomes, enabling the deletion of large genomic regions (up to 133 Kb) and replacement of sequences, thereby broadening the capabilities of genome engineering and simplifying applications in synthetic biology.
Implementation Method 1
allowing for targeted deletion or replacement of genomic regions by homologous recombination
Implementation Method 2
a guide crRNA complementary to a region of the prokaryotic target sequence
Data Source
AI summary
The present invention relates to kits and methods of modifying the prokaryotic genome a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system that utilized one nicking Cas nuclease and crRNAs. The kid and methods delete or replace portions of the prokaryotic genome. In some embodiments, an entire gene or multiple genes may be deleted or replaced.