Engineered Cpf1 Enzymes for Expanded PAM Recognition

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Solution Overview

Problem

Current genome-editing technologies, such as CRISPR-Cas systems, lack the ability to efficiently target multiple positions within the eukaryotic genome with diverse PAM recognition, necessitating the development of novel strategies for precise and affordable genome engineering.

Innovation Solution

Mutated Cpf1 polypeptides with altered PAM recognition capabilities are developed to expand the range of recognizable PAM sequences, allowing for more precise and versatile genome editing.

Engineering Contradictions & Design Principles

VSEngineering 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 it cannot target multiple positions within the eukaryotic genome with diverse PAM sequences

Engineering Contradiction:

ImprovePAM recognition diversityVSAvoidsystem complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent applies parameter changes by mutating specific amino acid residues in the Cpf1 protein sequence to alter PAM recognition specificity. Mutations at positions 130-177 (particularly 134, 135, 162-177) and 536-614 (particularly 542, 547-552, 607) change the biochemical properties of the PAM-interacting surface, enabling recognition of diverse PAM sequences including non-canonical variants with reduced T-content

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent achieves universality by engineering Cpf1 variants that can recognize multiple PAM sequence types. The mutated Cpf1 polypeptides maintain core endonuclease function while gaining expanded PAM recognition capabilities, allowing a single system to target multiple genomic positions with different PAM sequences

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Adaptability or versatility

If CRISPR-Cas systems are engineered to recognize diverse PAM sequences, then genome editing versatility improves, but the system requires more complex mutated polypeptide variants

Engineering Contradiction:

Improvegenome targeting capabilityVSAvoidpolypeptide production complexity

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The patent systematically changes amino acid parameters at specific positions to create discrete PAM recognition variants. Each mutation (e.g., at positions 134, 135, 162-177, 542, 547-552, 607) creates a defined variant with specific PAM preferences, enabling rational design and streamlined production of targeted polypeptide variants

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentUS12410416B2CRISPR enzymes and systems with modified pam specificity

Publication Date: 2025.09.09 THE BROAD INST INC

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.