Cpf1 PAM Specificity Engineering for Expanded Genome Targeting
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Solution Overview
Problem
Current genome-editing technologies, such as CRISPR-Cas9, have limitations in recognizing specific PAM sequences, limiting their versatility and efficiency in targeting multiple positions within the eukaryotic genome, necessitating the development of novel strategies for robust and affordable genome engineering.
Innovation Solution
Mutated Cpfl polypeptides with altered PAM recognition capabilities, allowing them to recognize a broader range of PAM sequences, including those shorter than 4 nucleotides, expanding the repertoire of PAM recognition and enhancing targeting specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR-Cas9 is used for genome editing, then precise genome targeting is achieved, but the ability to recognize diverse PAM sequences is limited
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the Cpfl polypeptide structure, particularly in the PAM recognition domain. These mutations alter the polypeptide's PAM specificity, enabling it to recognize diverse PAM sequences including non-canonical variants, thereby expanding the range of targetable genomic sites while maintaining editing precision
2Adaptability or versatility
If multiple genome positions are targeted, then comprehensive genome engineering is enabled, but the complexity of the system increases
Solution Approach 1:
The patent achieves universality by engineering Cpfl polypeptides with broad PAM recognition capabilities that can target multiple genome positions. The mutated Cpfl variants maintain a single modular structure (guid RNA + polypeptide) while gaining the ability to recognize diverse PAM sequences, eliminating the need for multiple specialized systems and simplifying the overall complexity
3Measurement precision
If PAM recognition specificity is increased, then targeting precision is improved, but the number of accessible target sites decreases
Solution Approach 1:
The patent resolves this contradiction by changing the PAM recognition parameters of the Cpfl polypeptide through targeted amino acid mutations. The mutated polypeptides maintain high specificity for their recognized PAM sequences while being able to recognize multiple different PAM types (including non-canonical variants), thereby increasing both the number of targetable sites and the overall versatility of the system
Data Source
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AI summary
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. Aspects of the invention in particular relate to Cpf1 mutants having altered PAM specificity.