Novel PAM Sequence Expands CRISPR Targeting Range

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Solution Overview

Problem

The CRISPR-Cas9 system for genome editing is limited by the availability of protospacer adjacent motifs (PAM) in target DNA regions, leading to insufficient on-target activity and off-target effects.

Innovation Solution

A novel PAM sequence is introduced to expand the range of targetable positions in the genome, allowing for efficient genome editing by combining a target nucleic acid, a CRISPR-associated (Cas) nuclease, and a guide RNA.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If the conventional CRISPR-Cas9 system with NGG PAM requirement is used, then the system is simple and well-established, but the targetable positions in the genome are limited

Engineering Contradiction:

Improvetargetable positions in genomeVSAvoidsystem complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent applies parameter changes by modifying the PAM sequence recognition requirements of the Cas9 nuclease. By engineering variants of SpCas9 that recognize alternative PAM sequences (such as NG, NRW, NGN), the system expands the range of targetable genomic positions without fundamentally changing the core CRISPR-Cas9 mechanism, thus improving adaptability while maintaining reasonable system complexity

Inventive Principle:

Principle #35Parameter changes

2Reliability

If the CRISPR-Cas9 system is applied to target DNA regions, then genome editing capability is achieved, but off-target effects occur

Engineering Contradiction:

Improveon-target activityVSAvoidoff-target effects

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent applies local quality by making the Cas9 nuclease selective for specific PAM sequence contexts. The engineered Cas9 variants maintain high activity at intended target sites with their specific PAM requirements while showing reduced activity at off-target sites, thereby improving on-target reliability and reducing off-target harmful effects through localized sequence specificity

Inventive Principle:

Principle #3Local quality

3Adaptability or versatility

If variants of CRISPR nucleases targeting non-NGG PAM sequences are developed, then the application range is broadened, but insufficient on-target activity remains

Engineering Contradiction:

ImprovePAM compatibilityVSAvoidon-target activity

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent addresses this contradiction by systematically engineering Cas9 variants with modified PAM recognition parameters. Through directed evolution and rational design, the patent optimizes both the PAM compatibility (expanding to NG, NRW, NGN sequences) and the catalytic activity parameters, achieving variants that maintain high on-target editing efficiency while recognizing diverse PAM sequences

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentUS12215315B2Method for modifying target nucleic acid in genome of cell

Publication Date: 2025.02.04 YONSEI UNIVERSITY BIOHEALTH TECHNOLOGY HOLDINGS INC

AI summary

Provided are: a method for modifying a target nucleic acid in the genome of a cell using a novel PAM sequence; and a cell in which a target nucleic acid in the genome is modified thereby. Accordingly, genome editing can be performed by targeting a position that could not be previously targeted as a target for genome editing, and thus the range of applications of genome editing can be expanded.