Engineered Cpf1 PAM Specificity for Broader Genome Targeting
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Solution Overview
Problem
Current genome-editing technologies, such as CRISPR-Cas systems, lack versatility in recognizing diverse PAM sequences, limiting their ability to target multiple positions within the eukaryotic genome efficiently and affordably.
Innovation Solution
Development of mutated Cpf1 polypeptides with altered PAM recognition specificity, allowing them to recognize different and/or shorter PAM sequences than wild-type Cpf1, expanding the repertoire of targetable sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR-Cas systems use wild-type Cpf1 with fixed PAM recognition, then the system is simple and easy to operate, but the ability to target multiple positions within the eukaryotic genome is limited
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the Cpf1 protein (particularly in the PAM-interacting regions) to alter PAM recognition specificity. This allows the same Cpf1 backbone to recognize different PAM sequences (e.g., changing from TTTV to TN or other variants), thereby expanding targetable genomic positions without requiring entirely different protein systems
Solution Approach 2:
The patent creates multi-functional Cpf1 variants that can recognize multiple different PAM sequences. By engineering Cpf1 mutants with relaxed or altered PAM requirements, a single guide RNA-Cpf1 complex can potentially target multiple genomic positions with different PAM contexts, increasing versatility while maintaining system simplicity
2Adaptability or versatility
If CRISPR-Cas systems are designed to recognize diverse PAM sequences, then the repertoire of targetable sites expands, but the precision and specificity of target recognition may be compromised
Solution Approach 1:
The patent applies local quality by making targeted amino acid substitutions specifically in the PAM-interacting regions of Cpf1 while leaving the guide RNA binding and target DNA recognition domains intact. This localized modification allows altered PAM recognition without compromising the precision of spacer sequence matching, maintaining target recognition accuracy while expanding PAM versatility
Solution Approach 2:
The patent creates Cpf1 variants with dynamic PAM recognition capabilities, where the protein can adapt its binding interface to recognize different PAM sequences based on the specific mutant configuration. This dynamic adaptability allows the system to maintain precise target recognition across diverse PAM contexts through engineered flexibility in the PAM-interacting regions
3Reliability
If traditional genome-editing technologies like zinc fingers or TALEs are used, then targeted genome perturbations can be achieved, but the systems are costly and difficult to set up and scale
Solution Approach 1:
The patent employs the CRISPR-Cas system as a simplified copy of genome-editing functionality compared to zinc finger or TALE technologies. By using programmable guide RNA sequences that direct Cpf1 to target sites, the system replaces complex protein-DNA recognition (required in zinc fingers and TALEs) with simpler RNA-DNA hybridization, making the system easier to design, manufacture, and scale while maintaining reliable targeted perturbation capability
Solution Approach 2:
The patent changes the fundamental recognition parameter from protein-DNA interaction (in zinc fingers/TALEs) to RNA-DNA interaction (in CRISPR-Cas). This parameter change simplifies the system architecture, reduces manufacturing complexity, and improves scalability while preserving the ability to achieve targeted genome perturbations through programmable guide sequences
Data Source
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.