3xNLS Cas9 Delivery for Consistent BCL11A Editing in HSCs

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Solution Overview

Problem

Existing CRISPR-Cas9 gene editing systems for altering BCL11A expression in hematopoietic stem cells have variable efficiency, specificity, and persistence, leading to potential genotoxicity and deleterious impacts on stem cell function.

Innovation Solution

Development of a CRISPR enzyme fused with multiple nuclear localization signal (NLS) sequences, specifically a c-Myc-like NLS at the amino terminus and SV40 and nucleoplasmin bipartate NLS at the carboxyl terminus, to enhance gene-editing efficacy in quiescent cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If CRISPR-Cas9 gene editing system is used to alter BCL11A expression in hematopoietic stem cells, then gene editing capability is achieved, but efficiency and specificity are variable leading to genotoxicity

Engineering Contradiction:

Improvegene editing efficiencyVSAvoidgenotoxicity

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent applies parameter changes by modifying the nuclear localization properties of the Cas9 enzyme through fusion with multiple NLS sequences. This changes the subcellular localization parameters of Cas9, increasing its concentration in the nucleus and thereby improving gene editing efficiency while reducing off-target effects and genotoxicity through more precise targeting

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent creates a composite protein structure by fusing Cas9 with multiple nuclear localization signal sequences from different sources (c-Myc-like NLS, SV40 NLS, and nucleoplasmin bipartate NLS). This composite enzyme combines the gene editing function of Cas9 with enhanced nuclear targeting capabilities, resulting in improved reliability and reduced harmful effects

Inventive Principle:

Principle #40Composite materials

2Duration of action of stationary object

If CRISPR-Cas9 system is used for gene modification, then gene editing capability is achieved, but persistence in hematopoietic stem cells is variable

Engineering Contradiction:

Improveediting persistenceVSAvoidediting consistency

Core Design Contradiction:

Duration of action of stationary objectVSReliability

Solution Approach 1:

The patent changes the localization parameter of Cas9 by adding multiple NLS sequences, which increases nuclear accumulation and residence time. This parameter modification leads to more persistent and consistent editing effects in hematopoietic stem cells, as the enzyme remains in the nucleus longer to complete editing events

Inventive Principle:

Principle #35Parameter changes

3Ease of manufacture

If Cas enzyme lacks NLS or has sub-optimal NLS composition, then delivery is simpler, but gene-editing efficacy in quiescent cells is reduced

Engineering Contradiction:

Improveenzyme delivery simplicityVSAvoidgene-editing efficacy

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The patent constructs a composite Cas9 enzyme with multiple NLS sequences fused to the Cas9 protein. This composite structure maintains relative simplicity in delivery (as it is still a single protein entity) while dramatically improving gene-editing efficacy in quiescent hematopoietic stem cells through enhanced nuclear import and retention

Inventive Principle:

Principle #40Composite materials

Data Source

PatentUS12522811B2Enhanced BCL11A RNP / CRISPR delivery and editing using a 3XNLS-CAS9

Publication Date: 2026.01.13 CHILDRENS MEDICAL CENT CORP

AI summary

Provided herein are synthetic nucleic acids that encode a CRISPR enzyme fused to a series of nuclear localization signal sequences for the use of altering expression of a gene in a cell. Further provided herein are methods for altering gene expression in a cell comprising introducing the synthetic nucleic acids and a guide RNA, or a polypeptide and a guide RNA into a cell. In certain embodiments the cell is a quiescent cell, for example, a hematopoietic stem cell.