Chimeric ScCas9-SpRY Enzyme for PAM-Free DNA Targeting

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Solution Overview

Problem

Current CRISPR-Cas systems require a specific protospacer adjacent motif (PAM) flanking the guide RNA-programmed target site, limiting the sequence accessibility for genome editing applications, particularly in therapeutically-relevant editing requiring precise genomic positioning.

Innovation Solution

The development of a chimeric SpRYc enzyme, combining the N-terminus of Sc++ with the PAM-interacting domain of SpRY, enables specific recognition and activity on DNA targets regardless of the downstream PAM sequence, allowing for 5′-NNN-3′ PAM preference.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If CRISPR-Cas systems use a specific PAM motif requirement, then DNA-protein interaction and cleavage efficiency are improved, but sequence accessibility and targetable genome locations are limited

Engineering Contradiction:

Improvecleavage efficiencyVSAvoidsequence accessibility

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent applies parameter changes by mutating specific amino acid residues in the PAM-interacting domain of Cas9 (particularly positions R1331, R1335, and T1337) to alter PAM recognition specificity. These point mutations change the biochemical parameters of PAM binding, enabling the enzyme to recognize diverse PAM sequences (5′-NNN-3′) while maintaining cleavage efficiency, thus resolving the contradiction between reliability and adaptability

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention creates a universal Cas9 variant (SpRYc) that can function across multiple target sequences regardless of specific PAM motifs. By engineering the PAM-interacting domain to accept any nucleotide combination (5′-NNN-3′), the enzyme achieves multi-functionality in genome editing applications, allowing targeting of any genomic location without being constrained by specific PAM requirements

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Measurement precision

If CRISPR-Cas systems require a PAM motif for target recognition, then cleavage specificity is improved, but genomic positioning flexibility is reduced

Engineering Contradiction:

Improvetarget recognition specificityVSAvoidgenomic positioning flexibility

Core Design Contradiction:

Measurement precisionVSEase of operation

Solution Approach 1:

The patent changes the recognition parameters of the PAM-interacting domain through rational mutagenesis, specifically modifying residues that contact PAM bases. This allows the enzyme to maintain specificity for the protospacer sequence while becoming insensitive to PAM variations, thereby improving genomic positioning flexibility without sacrificing target recognition precision

Inventive Principle:

Principle #35Parameter changes

3Reliability

If traditional Cas9 variants are used with strict PAM requirements, then off-target cleavage is reduced, but on-target accessibility at precise locations is limited

Engineering Contradiction:

Improvecleavage specificityVSAvoidtarget site accessibility

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The invention employs parameter changes in the PAM-interacting domain to decouple PAM recognition from cleavage activation. By mutating residues R1331, R1335, and T1337, the enzyme maintains high cleavage specificity for on-target sites while becoming accessible to any genomic location with a 5′-NNN-3′ PAM, thus simultaneously preserving reliability and enhancing adaptability

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent segments the Cas9 function into independent modules: the guide RNA binding domain maintains target specificity, while the engineered PAM-interacting domain provides flexible recognition. This functional segmentation allows the enzyme to distinguish between on-target and off-target sites through guide RNA complementarity while accepting diverse PAM sequences, resolving the contradiction between specificity and accessibility

Inventive Principle:

Principle #1Segmentation

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

SpRYc demonstrates high activity and accuracy on diverse 5′-NNN-3′ PAM sequences, enhancing genomic accessibility for therapeutic applications.

Implementation Method 1

CRISPR enzymes currently employed require a specified protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications

Methodology Applied

Scientific EffectDNA-protein interaction:

Implementation Method 2

SpRYc demonstrates high activity and accuracy on diverse 5′-NNN-3′ PAM sequences, enhancing genomic accessibility for therapeutic applications

Methodology Applied

Scientific EffectNuclease cleavage:

Data Source

PatentUS12529042B2Applications of recombined ScCas9 enzymes for PAM-free DNA modification

Publication Date: 2026.01.20 MASSACHUSETTS INST OF TECH

AI summary

SpRYc is a grafted ScCas9++-SpRY chimeric Cas9 possessing minimal 5′-NNN-3′ PAM specificity. SpRYc comprises the N-terminus (residues 1-1119) of ScCas9++ (Sc++), including the flexible loop, followed by the region of SpRY (residues 1111-1368) spanning its PAM-interacting domain mutations. Methods of altering gene expression include use of SpRYc in complex with guide RNA in a CRISPR-Cas9 system.