Cas12a PAM Recognition Variants for Expanded Genome Targeting

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Solution Overview

Problem

Existing CRISPR-Cas nucleases exhibit stringent protospacer adjacent motif (PAM) specificities, limiting the number of genomic target sites available for modification.

Innovation Solution

Development of CRISPR-Cas12a variants with altered PAM recognition specificity through targeted mutations at specific amino acid positions, enhancing the recognition of new PAMs and expanding the range of targetable genomic sites.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If stringent PAM specificity is maintained in CRISPR-Cas nucleases, then cutting precision is improved, but the number of available target sites is reduced

Engineering Contradiction:

Improvecutting precisionVSAvoidnumber of target sites

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The patent applies parameter changes by mutating specific amino acid residues in the PAM-interacting domain of Cas12a (such as positions K135, S168, T246, N263, T307, L324, E330, S345, R359, D367, I369, E379, Y381, I417, F474, E479, E484, D489, L498, and N590) to alter PAM recognition specificity. These mutations change the biochemical parameters of PAM binding, enabling the nuclease to recognize diverse PAM sequences (including TTTV, TTTV, TCR, and non-canonical PAMs) while maintaining cutting precision through controlled mutagenesis approaches

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If PAM recognition specificity is altered to recognize new PAMs, then versatility is improved, but recognition precision may be reduced

Engineering Contradiction:

ImprovePAM recognition rangeVSAvoidPAM recognition precision

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The patent applies local quality by making targeted mutations only in the PAM-interacting domain of Cas12a while leaving the rest of the protein structure intact. Specific amino acid positions (such as K135, S168, T246, N263, T307, L324, E330, S345, R359, D367, I369, E379, Y381, I417, F474, E479, E484, D489, L498, and N590) are mutated to confer new PAM specificities, while the overall protein architecture and guide RNA binding regions remain unchanged, thereby maintaining recognition precision through localized modification

Inventive Principle:

Principle #3Local quality

3Adaptability or versatility

If multiple mutations are introduced to expand PAM specificity, then adaptability is improved, but protein stability may be compromised

Engineering Contradiction:

ImprovePAM specificity diversityVSAvoidprotein stability

Core Design Contradiction:

Adaptability or versatilityVSStability of the object's composition

Solution Approach 1:

The patent applies partial action by introducing a limited number of targeted mutations (typically 1-5 key residues) in the PAM-interacting domain rather than extensive mutagenesis throughout the protein. This partial modification approach expands PAM specificity diversity while minimizing disruptions to the overall protein structure and stability, avoiding excessive mutations that could compromise structural integrity

Inventive Principle:

Principle #16Partial or excessive action

Data Source

PatentUS20260009010A1Methods and compositions for modification of protospacer adjacent motif specificity of CAS12a

Publication Date: 2026.01.08 PAIRWISE PLANTS SERVICES INC

AI summary

This invention relates to variants of Cas12a nucleases and engineered proteins 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.