Modified Cas12a Protein Enhances Genome Editing Specificity
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Solution Overview
Problem
The CRISPR-Cas system based on the Cpf1 protein faces off-target problems, leading to non-target DNA cleavage and mutations in undesired genes, which complicates its application in research and medical fields, and there is a need for genetic scissors with excellent indel efficiency that operate specifically at the target site without off-target effects.
Innovation Solution
A modified Cas12a protein with myc-NLS linked to its C-terminus, combined with a guide RNA and an enhancer, is used to enhance the genome editing efficiency by improving the precision and accuracy of DNA cleavage, reducing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional Cas9 or Cpf1-based CRISPR-Cas systems are used for genome editing, then genome editing capability is achieved, but off-target effects occur leading to non-target DNA cleavage and mutations
Solution Approach 1:
The patent modifies the Cpf1 protein by changing specific amino acid residues (e.g., R157A, R157K, R157R, R157H, R157Q, R157E substitutions) to alter its biochemical properties and reduce off-target binding affinity while maintaining on-target cleavage activity. This parameter change in the protein sequence directly addresses the specificity problem.
Solution Approach 2:
The patent creates modified versions of the Cpf1 protein by copying the original sequence and introducing specific mutations. These copied and modified protein variants (e.g., mgCas12a variants) are designed to have improved specificity compared to the wild-type Cpf1, thereby reducing off-target effects while preserving genome editing function.
2Productivity
If modified Cas12a protein with myc-NLS is used, then nuclear localization and endonuclease activity are improved, but protein structure complexity increases
Solution Approach 1:
The myc-NLS peptide sequence acts as an intermediary element that mediates nuclear localization of the Cas12a protein. By adding this short peptide tag (myc-NLS: EQKLISEEDL), the protein gains improved nuclear import capability without fundamentally altering its core catalytic structure, thus enhancing productivity with minimal structural complexity increase.
Solution Approach 2:
The modified Cas12a protein is segmented into functional domains: the core Cas12a catalytic domain and the appended myc-NLS localization signal. This segmentation allows independent optimization of each function - the core domain maintains catalytic activity while the added segment provides enhanced nuclear localization, improving overall genome editing efficiency.
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 modified Cas12a protein demonstrates significantly improved endonuclease activity and indel efficiency, effectively utilizing the CRISPR-Cas system for precise genome editing, achieving higher knockout efficiencies compared to conventional systems, especially when used with an enhancer.
Implementation Method 1
When the Cas protein forms a complex with two RNAs, termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), it forms an active endonuclease, thereby cleaving foreign genetic elements
Implementation Method 2
myc-NLS (nuclear localization sequences) comprising the amino acid sequence of SEQ ID NO: 4 linked to the C-terminus of the Cas12a protein
Data Source
AI summary
The present invention relates to a modified CRISPR-associated protein and a use thereof. More specifically, the present invention relates to a composition for genome editing comprising the modified CRISPR-associated protein and an enhancer, a method for genome editing using the same, and a method for producing a transformant by using the same. The modified CRISPR-associated protein according to the present invention is available as a nuclease having excellent indel efficiency in a CRISPR-Cas system, and exhibits excellent indel efficiency compared to conventional CRISPR-Cas systems, thus finding advantageous applications in genome editing.