Cas12a Nuclease PAM Engineering for Broader Genomic Targeting

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Solution Overview

Problem

Existing CRISPR-Cas nucleases exhibit stringent PAM specificities that limit the number of genomic target sites available for modification, reducing their usefulness in genome editing.

Innovation Solution

Development of modified CRISPR-Cas12a nucleases with altered PAM recognition specificities, including mutations at specific positions in the Cas12a polypeptide, and engineered proteins with heterologous components, to recognize a broader range of PAM sequences, enabling more efficient genome editing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If stringent PAM specificity is maintained in CRISPR-Cas nucleases, then nuclease activity and precision are improved, but the number of available genomic target sites is reduced

Engineering Contradiction:

ImprovePAM recognition precisionVSAvoidnumber of target sites

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The patent applies parameter changes by systematically mutating amino acid residues in the PAM-interacting region of Cas12a (specifically positions 532, 538, 542, 595, 680, 690, and 743) to alter PAM recognition specificity. This allows the nuclease to recognize diverse PAM sequences beyond the native TTTV motif, thereby increasing the number of available target sites while maintaining nuclease activity

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent creates engineered Cas12a variants with broadened PAM recognition capabilities that can function with multiple different PAM sequences (including TTTV, TTAA, TTAC, ACCA, ACCC, ACCG, and many others). This multi-functionality allows a single nuclease construct to target a much larger portion of the genome, resolving the contradiction between precision and versatility

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Adaptability or versatility

If Cas12a variants with relaxed PAM specificity are created, then the number of targetable sites increases, but nuclease activity may be reduced

Engineering Contradiction:

Improvenumber of target sitesVSAvoidnuclease activity

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent systematically tests and optimizes amino acid substitutions at PAM-interacting residues to find mutations that broaden PAM recognition while preserving catalytic activity. Specific mutation combinations (such as R532R/K595R, R532R/K538V/Y542R, and R680R/R690V) have been identified that maintain high nuclease activity across diverse PAM sequences

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs iterative testing and characterization of mutant variants to identify those that successfully balance broad PAM recognition with maintained nuclease activity. Through systematic evaluation of cutting efficiency across multiple PAM types, optimal mutation combinations are selected that preserve reliable nuclease function while expanding target site availability

Inventive Principle:

Principle #23Feedback

Data Source

PatentUS12428636B1Methods and compositions for modification of protospacer adjacent motif specificity of CAS12A

Publication Date: 2025.09.30 PAIRWISE PLANTS SERVICES INC

AI summary

This invention relates to variants of Cas12a nucleases having altered protospacer adjacent motif recognition specificity. The invention further relates to methods of making CRISPR-CAS nuclease variants and methods of modifying nucleic acids using the variants.