Cas12a Protein Variant Mutations for Lower Off-Target Gene Editing

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Solution Overview

Problem

Current gene editing technologies, particularly those based on CRISPR-Cas12a, suffer from off-target effects due to trans-cleavage activity, leading to cytotoxicity issues.

Innovation Solution

A gene editing protein variant with mutations at specific core amino acid sites, such as F1081 and/or K1069 of FnCas12a, is developed to reduce trans-cleavage activity by ≥50%, thereby minimizing off-target effects and cytotoxicity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If Cas12a exhibits trans-cleavage activity towards single-stranded DNA, then the protein can process various DNA forms, but off-target effects and cytotoxicity increase

Engineering Contradiction:

ImproveDNA processing capabilityVSAvoidoff-target effects and cytotoxicity

Core Design Contradiction:

Adaptability or versatilityVSObject-affected harmful factors

Solution Approach 1:

The patent applies parameter changes by mutating specific amino acid residues (F1081 and/or K1069) in the Cas12a protein sequence. These mutations alter the protein's biochemical properties to eliminate trans-cleavage activity while preserving cis-cleavage activity, thereby resolving the contradiction between DNA processing capability and harmful off-target effects

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent applies local quality by making targeted changes at specific positions (1081 and/or 1069) in the protein sequence rather than global modifications. This localized approach allows the protein to maintain its overall structure and cis-cleavage function while specifically removing the harmful trans-cleavage capability at the affected residues

Inventive Principle:

Principle #3Local quality

2Reliability

If wild-type Cas12a is used for gene editing, then cis-cleavage activity is achieved, but trans-cleavage activity causes off-target cleavage of single-stranded DNA

Engineering Contradiction:

Improvecis-cleavage specificityVSAvoidtrans-cleavage off-target effects

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The patent converts the harmful trans-cleavage activity into a beneficial feature by using the same protein framework (Cas12a) that naturally possesses both activities, then selectively eliminating the harmful trans-cleavage function through mutation while preserving the useful cis-cleavage function, effectively turning a harmful characteristic into a controlled feature

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Data Source

PatentUS20260028605A1Gene editing protein variant capable of reducing gene editing off-target rate

Publication Date: 2026.01.29 SHANGHAI TOLO BIOTECH CO LTD

AI summary

A gene editing protein variant is capable of reducing a gene editing off-target rate. The variant is an unnatural protein with cis-cleavage activity, and the variant has reduced trans-cleavage activity as compared to a wild-type gene editing protein thereof. Furthermore, the variant is mutated at one or more of cleavage activity-related core amino acid sites of the wild-type gene editing protein selected from the following: a phenylalanine (F) site corresponding to the 1081st position of FnCas12a; and/or a lysine (K) site corresponding to the 1069th site of the FnCas12a. The variant can have cis-cleavage activity and reduced trans-cleavage activity. Moreover, the gene editing protein variant or a gene editing system having the gene editing protein variant can significantly reduce the gene editing off-target rate.