Mutant Cpf1 Endonucleases for Single-Strand Cleavage
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Solution Overview
Problem
Current CRISPR endonucleases, such as Cas9 and Cpf1, are used primarily in their natural forms, limiting their potential applications and efficiency in introducing specific or non-specific nucleic acid breaks.
Innovation Solution
Development of mutant Cpf1 endonucleases with altered activity, capable of introducing single strand breaks or double strand breaks in nucleic acid target sequences, both specifically and non-specifically, and forming crRNA-Cpf1 complexes for enhanced target recognition and cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If wild type Cpf1 endonuclease is used, then specific DNA cleavage activity is maintained, but versatility in different cleavage modes (single strand vs double strand breaks, specific vs non-specific binding) is limited
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at positions 918, 1013, 1014, 1025, and 1028 of the Cpf1 protein sequence. These parameter changes in the protein structure enable the endonuclease to exhibit different cleavage activities including single strand breaks, double strand breaks, and non-specific binding modes while maintaining controllable specificity through crRNA guide design
2Productivity
If mutant Cpf1 endonucleases with altered activity are developed, then versatility and detection efficiency are improved, but protein structure complexity increases
Solution Approach 1:
The patent improves productivity by optimizing specific amino acid parameters at key positions (918, 1013, 1014, 1025, 1028) to enhance detection efficiency and cleavage activity. These targeted parameter changes achieve high productivity without requiring complete structural redesign, thus limiting the increase in overall protein structure complexity
Solution Approach 2:
The patent applies local quality by introducing mutations only at specific functional domains (positions 918, 1013, 1014, 1025, 1028) rather than throughout the entire protein structure. This localized approach improves detection efficiency and versatility while minimizing changes to the overall protein structure and maintaining structural integrity
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 mutant Cpf1 endonucleases demonstrate improved efficiency in detecting and quantifying nucleic acid sequences, even at low concentrations, and are suitable for diagnostic applications, such as detecting infectious diseases.
Implementation Method 1
Cpf1 forms with a relatively small (between 40 to 45 bases) crRNA (CRISPR-RNA) molecule, a ribonucleoprotein complex that is able to recognize, unwind and cut with high specificity the DNA-target complementary to the crRNA
Implementation Method 2
Cpf1 is a DNA endonuclease, belonging to the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats) class 2 type V-B adaptive immune system
Data Source
AI summary
The present invention relates to mutant Cpf1 endonucleases having altered activity compared to the wild type Cpf1, and their use to introduce single strand breaks in nucleic acid sequences. Methods for detection and quantification of a nucleic acid sequence are also disclosed. Methods for diagnosis of an infectious disease are also disclosed.