Modified Pre-crRNA for Efficient and Allele-Specific Cas3 Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The class-1 CRISPR-Cas system faces challenges in achieving stable and efficient genome editing in cells with low gene transfer efficiency, and there is a need for methods to accurately analyze genome editing patterns and specifically edit one allele, particularly in the context of genetic diseases like myotonic dystrophy I.
Innovation Solution
Introduce chemical modifications to the pre-crRNA for the CRISPR-Cas3 system, particularly in specific regions, and develop tools like SSA reporter vectors and droplet digital PCR (ddPCR) to enhance editing efficiency and pattern analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical modifications are introduced to pre-crRNA to improve stability and editing efficiency, then genome editing efficiency is enhanced, but the complexity of pre-crRNA design and synthesis increases
Solution Approach 1:
The patent applies chemical modifications (2'-O-methyl and phosphorothioate) specifically at the 5' and 3' terminal regions of pre-crRNA, rather than throughout the entire molecule. This localized approach enhances stability and editing efficiency while minimizing synthesis complexity and cost compared to full-length modification.
Solution Approach 2:
The patent systematically varies parameters including modification type (2'-O-methyl, phosphorothioate), modification position (5' terminal, 3' terminal), and number of modified nucleotides (1-5 nucleotides per terminal) to optimize genome editing efficiency while maintaining manageable synthesis complexity.
2Productivity
If modified nucleotides are used in pre-crRNA to enhance editing efficiency, then cleavage activity is improved, but the difficulty of synthesizing pre-crRNA increases
Solution Approach 1:
Chemical modifications are concentrated at the terminal regions (5' and 3' ends) where they provide maximum benefit for stability and Cas3 recruitment, while leaving the central spacer and repeat regions unmodified to simplify synthesis and reduce costs.
Solution Approach 2:
The patent uses a limited number of modified nucleotides (1-5 per terminal) rather than full modification, which provides sufficient enhancement of cleavage activity while keeping synthesis complexity and cost at acceptable levels.
3Reliability
If the recognition sequence of guide RNA is made longer to improve specificity, then off-target effects are reduced, but the difficulty of designing specific guides increases
Solution Approach 1:
The patent exploits the asymmetric structure of pre-crRNA, with distinct 5' and 3' terminal regions containing repeat sequences and a central spacer region. Chemical modifications are applied asymmetrically at the terminals, enhancing specificity through improved Cas3 recruitment without requiring extended recognition sequences.
Solution Approach 2:
The patent optimizes the length and composition of the spacer sequence (typically 20-50 nucleotides) to achieve high specificity for the target gene, balancing recognition specificity with design simplicity.
Data Source
Figure 1~2
Figure 3-1
Figure 3-2A~3-2F
AI summary
The present invention is a pre-crRNA of a type I CRISPR-Cas system having a modified nucleotide, wherein, in a region consisting of the 5' arm region of the first repeat sequence, the 5'-side stem-forming region of the first repeat sequence, and the loop-forming region of the first repeat sequence, at least one nucleotide is the modified nucleotide.