BCL11A CRISPR RNP Editing with 3xNLS-Cas9 Nuclear Delivery
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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, enhancing gene-editing efficacy in hematopoietic cells.
Engineering Contradictions & Design Principles
Engineering 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 editing efficiency, specificity, and persistence are variable leading to potential genotoxicity and stem cell dysfunction
Solution Approach 1:
The patent introduces nuclear localization signals (NLS) as intermediary sequences that mediate the transport of Cas9 enzyme into the nucleus. By adding specific NLS sequences (such as SV40 NLS, nucleoplasmin NLS, or c-Myc NLS) to the Cas9 protein, the system achieves more reliable nuclear entry and subsequent gene editing, while reducing off-target effects and genotoxicity through improved specificity.
2Productivity
If CRISPR-Cas9 system is used for gene editing in hematopoietic stem cells, then gene modification is achieved, but editing efficiency and persistence are variable
Solution Approach 1:
The patent modifies the Cas9 protein by changing parameters such as adding or modifying nuclear localization signals. This parameter change enhances the editing efficiency by improving nuclear entry and maintains persistence by ensuring sustained Cas9 activity in the nucleus, leading to more durable gene editing outcomes in hematopoietic stem cells.
3Reliability
If CRISPR-Cas9 system is used to decrease BCL11A expression, then fetal hemoglobin induction is achieved, but variable efficiency and specificity lead to potential deleterious impacts
Solution Approach 1:
The patent uses nuclear localization signals as intermediaries to enhance the reliability of BCL11A expression decrease and subsequent fetal hemoglobin induction. The NLS sequences mediate precise nuclear delivery of Cas9, improving specificity and reducing off-target effects that could cause deleterious impacts on stem cell function.
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
The CRISPR enzyme with multiple NLS sequences demonstrates improved gene-editing capability, reducing BCL11A expression effectively and safely in hematopoietic cells, promoting fetal hemoglobin induction without detectable genotoxicity or stem cell dysfunction.
Implementation Method 1
CRISPR enzymes fused to nuclear localization signal (NLS) sequences, where the number and type of NLS sequences increases its gene-editing efficacy
Data Source
Figure 1A~1D
Figure 1E~1I
Figure 1J~2A
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.