Cas12a Endonuclease Engineering for High-Precision Genome Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR technology suffers from low precision in genome editing due to traditional endonucleases, necessitating the development of novel, highly efficient endonucleases for targeted genetic modifications.
Innovation Solution
A composition comprising a type V CRISPR-associated protein, such as Cas12a, with specific amino acid sequences and a guide RNA that is reverse complementary to eukaryotic nucleic acid sequences, enhancing genome editing efficiency by at least two-fold compared to existing Cas12a orthologs like AsCas12a, FnCas12a, or LbCas12a.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional endonucleases are used in CRISPR technology, then genome editing can be performed, but precision is relatively low
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the Cas12a protein sequence (particularly in the PAM-interacting region) to optimize binding affinity and specificity. This involves changing chemical parameters of the endonuclease to achieve both high precision targeting and reliable editing outcomes, resolving the contradiction between precision and reliability.
Solution Approach 2:
The patent applies local quality by making targeted modifications to specific regions of the Cas12a protein (such as residues 829-991 or 825-996) while maintaining the overall protein structure. This localized optimization of the PAM-interacting region enhances precision without compromising the overall reliability of the genome editing system.
2Productivity
If Cas12a proteins with high editing efficiency are developed, then genome editing efficiency increases, but protein sequence complexity increases
Solution Approach 1:
The patent optimizes specific parameters of the Cas12a protein (amino acid sequences in regions 829-991 or 825-996) to achieve at least 2-fold increased editing efficiency. By focusing changes on specific parameters rather than redesigning the entire protein, the solution increases productivity while minimizing the addition of overall complexity.
Solution Approach 2:
The patent performs preliminary computational design and in silico screening of Cas12a variants before experimental validation. This preliminary action identifies optimal amino acid substitutions that enhance efficiency, reducing the need for extensive trial-and-error experimentation and thereby managing complexity while achieving high productivity.
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
The composition exhibits improved genome editing efficiency, specifically cleaving eukaryotic nucleic acid sequences with increased precision and efficiency, particularly targeting regions like KRAS, HER2/neu, and other cancer-related genes, thereby facilitating effective genetic modifications.
Implementation Method 1
a guide RNA that is reverse complementary to eukaryotic nucleic acid sequences
Implementation Method 2
type V CRISPR-associated protein, such as Cas12a, with specific amino acid sequences... cleaving eukaryotic nucleic acid sequences
Data Source
AI summary
The present disclosure provides novel Cas12a proteins, which cleave target nucleic acids and methods of use thereof.