Cas12a PAM-Recognition Mutations for Higher DNA Cleavage

Solution Overview

Problem

The Cas12a system has limited utility due to its low enzymatic activity and restricted PAM compatibility, making it less efficient for genome editing compared to Cas9, particularly at AT-rich sites, and requires improvement for broader application in eukaryotic cells.

Innovation Solution

Development of novel AsCpf1 and AsCas12a variants, such as M537R, F870L, and their combinations, which enhance DNA cleavage activity at both canonical and non-canonical PAM sites, and include nuclear localization signals for efficient delivery to eukaryotic cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If Cas12a is used for genome editing, then AT-rich sites can be targeted that are inaccessible to Cas9, but the enzymatic activity is relatively low reducing editing efficiency

Engineering Contradiction:

ImprovePAM compatibilityVSAvoidenzymatic activity

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent applies parameter changes by mutating specific amino acid residues in the Cas12a protein structure (e.g., positions 537, 870, 510, 569, 599, 638) to alter the PAM recognition parameters. These mutations enable the enzyme to recognize both canonical TTTV PAM sequences and non-canonical PAM sequences (such as TTTC, TTTG, TTTC, TTCA), thereby expanding targetable sites while maintaining or enhancing enzymatic activity through optimized cleavage efficiency at these diverse PAM types.

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If Cas12a is used instead of Cas9, then targetable loci are expanded particularly at AT-rich sites, but the likelihood of efficient genome editing is much lower

Engineering Contradiction:

Improvetargetable lociVSAvoidgenome editing efficiency

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent applies parameter changes by mutating specific amino acid residues in the Cas12a protein structure (e.g., positions 537, 870, 510, 569, 599, 638) to alter the PAM recognition parameters. These mutations enable the enzyme to recognize both canonical TTTV PAM sequences and non-canonical PAM sequences (such as TTTC, TTTG, TTTC, TTCA), thereby expanding targetable sites while maintaining or enhancing enzymatic activity through optimized cleavage efficiency at these diverse PAM types.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent creates composite CRISPR systems by combining mutated Cas12a proteins with specifically designed crRNAs that target diverse PAM sequences. This composite approach integrates the expanded PAM recognition capability of the mutated Cas12a with optimized guide RNA molecules, resulting in a synergistic system that achieves high editing efficiency across a broader range of genomic sites including AT-rich regions that were previously inaccessible or poorly targetable.

Inventive Principle:

Principle #40Composite materials

3Productivity

If novel mutations are introduced to enhance DNA cleavage activity, then cleavage efficiency at TTTT PAM sites is improved, but protein structure and function may be disrupted

Engineering Contradiction:

ImproveDNA cleavage efficiencyVSAvoidprotein structure

Core Design Contradiction:

ProductivityVSStability of the object's composition

Solution Approach 1:

The patent applies local quality by introducing mutations at specific, localized positions within the Cas12a protein structure (e.g., residues at positions 537, 870, 510, 569, 599, 638) that are strategically located in regions responsible for PAM recognition and DNA binding. These localized mutations enhance DNA cleavage efficiency at TTTT and other non-canonical PAM sites while preserving the overall protein structure and function by avoiding disruptions to critical catalytic domains and structural frameworks.

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS20250346876A1Novel Mutations That Enhance the DNA Cleavage Activity of Acidaminococcus Sp. CPF1

Publication Date: 2025.11.13 INTEGRATED DNA TECHNOLOGIES INC

AI summary

The present disclosure concerns polynucleotides and amino acids of Acidaminococcus sp. Cas12a (Cpf1) and methods for their use for genome editing in eukaryotic cells.