SaCas9 N413 Mutation Reduces Off-Target Cleavage

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Solution Overview

Problem

CRISPR-Cas9 systems, such as Streptococcus aureus Cas9 (SaCas9), face limitations due to off-target activities that lead to undesired mutations, restricting their broad application in genome editing and gene therapy.

Innovation Solution

A modified SaCas9 protein with mutations at specific positions, including N413, R245, N419, and R654, is developed to reduce nuclease activity at off-target sites, enhancing genome-wide specificity and editing efficiency when used with a guide RNA.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If wild-type SaCas9 protein is used for genome editing, then editing efficiency is achieved, but off-target activity occurs leading to undesired mutations

Engineering Contradiction:

Improvegenome editing precisionVSAvoidoff-target activity

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The patent applies parameter changes by introducing specific amino acid substitutions at positions N413, R245, N419, and R654 in the SaCas9 protein sequence. These parameter changes in the protein structure modify the nuclease activity characteristics, reducing off-target cleavage while preserving on-target editing efficiency. The systematic variation of amino acid parameters resolves the contradiction between reliability and harmful off-target effects.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent applies local quality by making targeted modifications at specific positions (N413, R245, N419, R654) within the SaCas9 protein rather than changing the entire protein. These localized quality changes at critical residues affect the protein's interaction with off-target DNA sequences while maintaining its overall function at the intended target site, thus resolving the contradiction between precision and off-target activity.

Inventive Principle:

Principle #3Local quality

2Productivity

If SaCas9 protein is used to target genomic loci, then genome editing capability is achieved, but non-perfect guide-RNA-target-DNA matching leads to modifications at unintended loci

Engineering Contradiction:

Improvegenome editing capabilityVSAvoidgenomic locus specificity

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The patent changes the biochemical parameters of the SaCas9 protein by substituting amino acids at key positions, which alters the protein's DNA binding characteristics and cleavage specificity. This parameter modification enables the protein to distinguish more effectively between perfect and non-perfect guide-RNA-target-DNA matches, thereby improving manufacturing precision while maintaining productivity.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent replaces the wild-type SaCas9 protein's natural DNA recognition mechanism with a modified version that has altered biochemical properties. The substituted amino acids change the mechanical and chemical interactions between the protein and DNA, creating a more selective system that reduces off-target modifications while preserving on-target editing capability.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Data Source

PatentUS10975364B2Modified protein and method for altering genome of cell

Publication Date: 2021.04.13 CITY UNIVERSITY OF HONG KONG

AI summary

A modified Streptococcus aureus Cas9 (SaCas9) protein with a mutation at an N413 position, and optionally one or more of a nuclear localization sequence, a cell penetrating peptide sequence, an affinity tag and/or a fusion base editor protein, and a kit that comprises said modified protein. A method for altering the genome of a cell, the method including the step of using the modified protein of the invention.