Engineered Type V CRISPR Nickase for Target-Strand Editing
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Solution Overview
Problem
There is no clear way to produce a Type V CRISPR target strand nickase due to structural and mechanistic differences with Type II CRISPR endonucleases, and mutating the catalytic residue of the RuvC domain in Type V CRISPR endonucleases results in a deactivated enzyme rather than a target strand nickase.
Innovation Solution
Engineered proteins comprising a Type V CRISPR-Cas effector polypeptide devoid of a nuclease domain, combined with a second polypeptide having specific amino acid sequences, to create a functional target strand nickase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the catalytic residue of the RuvC domain is mutated in Type V CRISPR endonucleases, then nuclease activity is prevented, but the enzyme becomes completely deactivated rather than functioning as a target strand nickase
Solution Approach 1:
The patent divides the RuvC domain into functionally distinct regions: the catalytic residue (mutated to prevent double-strand cleavage) and the nontarget strand binding/cleavage region (intact to enable nickase activity). This segmentation allows selective preservation of target strand nicking capability while eliminating harmful double-strand break activity.
Solution Approach 2:
The patent applies local quality by making the catalytic residue specifically mutant (e.g., D1003A in LbCas12a) while keeping the rest of the RuvC domain wild-type. This localized modification creates a nickase with asymmetric functionality: the mutant catalytic site cannot perform full cleavage, but the intact nontarget strand binding region enables selective nicking of the target strand.
2Manufacturing precision
If Type V CRISPR endonucleases are used for genome editing, then staggered double-strand breaks are created, but this mechanism lacks the precision and control of target strand nicking
Solution Approach 1:
The patent extracts the target strand cleavage function from the Type V CRISPR endonuclease by mutating the catalytic residue of the RuvC domain. This extraction converts the enzyme from a double-strand breaker to a single-strand nicker, providing precise control over the editing outcome while simplifying the mechanism to match Type II CRISPR nickase behavior.
3Adaptability or versatility
If engineered proteins are constructed with Type V CRISPR-Cas effector polypeptides devoid of nuclease domain combined with second polypeptides, then target strand nickase function is achieved, but protein structure complexity increases
Solution Approach 1:
The patent merges the Type V CRISPR-Cas effector polypeptide (with mutated catalytic residue) and the second polypeptide (SEQ ID NOs: 176-207) into a single engineered protein. This combination creates a functional nickase where the effector domain provides target recognition and binding, while the second polypeptide contributes to stable complex formation and enhanced nickase activity.
Data Source
AI summary
Described herein are engineered proteins and methods of use of such proteins. Also described herein are complexes, compositions, and systems including engineered proteins of the present invention, each of which may be used for modifying and/or editing a target nucleic acid. An engineered protein of the present invention may be an enzyme and/or may be an RNA-guided DNA-binding protein.