SpCas9 Variant Mutations Expand PAM Recognition Range

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Solution Overview

Problem

The existing SpCas9 protein is limited to recognizing and cleaving double-stranded DNA adjacent to the specific PAM sequence 5′-NGG-3′, restricting gene editing to specific sites.

Innovation Solution

A SpCas9 variant with specific mutations, such as L1111R/D1135V/G1218K/E1219V/A1322R/R1335Q, is developed to recognize and cleave DNA at PAM sequences other than 5′-NGG-3′, including 5′-NGN-3′ and 5′-NNG-3′, and potentially be PAMless.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If the wild-type SpCas9 protein is used, then the PAM recognition is specific (5'-NGG-3'), but the gene editing site range is limited

Engineering Contradiction:

ImprovePAM sequence recognition rangeVSAvoidPAM recognition specificity

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent applies parameter changes by mutating specific amino acid residues in the SpCas9 protein structure, particularly in the PAM recognition domain. The mutations (e.g., G1218, E1219, R1333, R1335, T1337) alter the chemical and structural parameters of the PAM recognition interface, enabling the protein to recognize diverse PAM sequences including 5'-NGG-3', 5'-NAG-3', 5'-NAC-3', and potentially PAMless sequences, thus expanding the gene editing site range while maintaining functional reliability

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If the SpCas9 protein is engineered to recognize various PAM sequences, then the gene editing site selection range expands, but the protein structure complexity increases

Engineering Contradiction:

ImproveGene editing site rangeVSAvoidProtein structure complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent applies local quality by introducing mutations only in specific regions of the SpCas9 protein structure that are directly involved in PAM recognition (residues 1218-1219 and 1333-1337), while leaving the rest of the protein structure unchanged. This localized modification approach enables diverse PAM recognition without unnecessarily increasing overall protein structure complexity

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent creates a universal SpCas9 variant that can perform multiple PAM recognition functions through a single engineered protein structure. The mutated Cas9 protein serves as a multi-functional enzyme capable of recognizing various PAM sequences (5'-NGG-3', 5'-NAG-3’, 5'-NAC-3’, and potentially PAMless), eliminating the need for multiple different Cas9 proteins for different editing sites

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS20250154479A1Streptococcus pyogenes-derived cas9 variant

Publication Date: 2025.05.15 TOOLGEN INC

AI summary

The present disclosure discloses a SpCas9 variant. The SpCas9 variant is characterized by the ability to recognize a PAM sequence different from that of a wild-type SpCas9 protein.