Guide RNA Termination Sequences for Precise CRISPR Gene Control
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Solution Overview
Problem
Existing methods for regulating gene expression or activity using CRISPR/Cas systems are limited by inefficient termination of guide nucleic acid molecules, leading to unwanted off-target effects and reduced specificity.
Innovation Solution
Incorporation of polyX sequences, specifically polyT sequences, within the guide nucleic acid molecules to enhance termination efficiency and reduce expression, thereby improving the regulation of target gene activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR/Cas technology is used to manipulate polynucleotide sequences, then versatility and programmability are improved, but off-target effects increase and specificity decreases
Solution Approach 1:
The patent incorporates termination sequences (polyT in DNA, polyU in RNA) at specific positions within the guide nucleic acid molecule before transcription/translation occurs. This preliminary action ensures that the guide RNA is truncated to a specific length, preventing it from binding to off-target sites while maintaining on-target specificity. The termination sequence acts in advance to define the functional boundaries of the guide RNA.
Solution Approach 2:
The guide nucleic acid molecule is segmented into distinct functional domains: a 5' domain containing the termination sequence, a middle domain containing the target-binding sequence, and a 3' domain. This segmentation allows the target-binding domain to be optimized for specificity while the 5' domain controls the overall length and prevents off-target effects through premature transcription termination.
2Adaptability or versatility
If guide nucleic acid molecules are used to regulate target genes, then gene regulation capability is improved, but expression control precision deteriorates
Solution Approach 1:
The termination sequence is incorporated into the polynucleotide encoding the guide nucleic acid molecule before expression occurs. This preliminary structural element ensures that transcription terminates at a defined position, producing a guide RNA of precise length. This pre-determined structure enables precise control over which target sites are regulated, improving expression control precision.
3Object-affected harmful factors
If polyX sequences are incorporated to reduce guide nucleic acid expression, then off-target effects are reduced, but termination efficiency worsens without optimal sequence design
Solution Approach 1:
The patent optimizes specific parameters of the polyX sequence: using polyT (DNA) or polyU (RNA) with a specific length range (5-20 nucleotides), positioning it at the 5' end of the guide nucleic acid encoding sequence, and ensuring it is not part of the terminal domain. These parameter changes ensure high termination efficiency while maintaining the ability to reduce off-target effects.
Solution Approach 2:
The termination sequence is placed locally at a specific position (5' end, non-terminal) rather than uniformly throughout the molecule. This local placement creates a specific functional zone that controls transcription termination without interfering with the target-binding capability of the rest of the guide RNA, ensuring both high termination efficiency and reduced off-target effects.
Data Source
AI summary
Provided herein are systems of regulating expression of a cargo (e.g., a guide nucleic acid) from a polynucleotide sequence (e.g., a vector).