Single AAV CRISPRi System for Gene Modulation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR-based systems face challenges in delivering functional CRISPR machinery due to size constraints of adeno-associated virus (AAV) vectors, requiring multiple vectors for efficient gene modulation, which is inefficient and unsafe, especially for transducing neurons, and struggles with modulating endogenous gene expression effectively.
Innovation Solution
A novel CRISPRi system is designed to fit within a single AAV vector by optimizing the size and components, including a nucleic acid molecule encoding a Cas polypeptide with a repressor domain, a nuclear localization signal, and a guide RNA, allowing for efficient nuclear targeting and gene expression modulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple AAV vectors are used to deliver functional CRISPR machinery, then gene modulation efficiency is improved, but transduction efficiency and safety deteriorate
Solution Approach 1:
The patent combines SadCas9 and gRNA into a single AAV vector by optimizing the construct size to fit within the 4.7 kb packaging limit. This merging eliminates the need for dual AAV transduction, improving safety and transduction efficiency while maintaining gene modulation functionality through the integrated CRISPRi system
2Device complexity
If AAV packaging size constraint is reduced to fit single vector, then device complexity is reduced, but manufacturing precision deteriorates
Solution Approach 1:
The patent changes the parameter of Cas9 protein by selecting SadCas9 (Staphylococcus aureus Cas9) which has a smaller size (3.2 kb) compared to other Cas9 variants. This parameter change enables fitting the complete CRISPRi system including promoter, gRNA, and poly(A) regions within the AAV packaging limit of 4.7 kb, simplifying the vector system while requiring precise construct design
Data Source
AI summary
A nucleic acid molecule is uniquely designed and encodes an entire CRISPRi or CRISPRa system, while being sized for packaging within a single adeno-associated virus (AAV) vector. Examples of the nucleic acid molecule include about 4600 to 4700 base pairs. Examples of the nucleic acid molecule can include a nucleotide encoding a Cas polypeptide; a nucleotide encoding a repressor or an activator domain attached to the nucleotide encoding the Cas polypeptide via a linker; a first promoter operably connected to the nucleotide encoding the repressor or activator domain or the nucleotide encoding the Cas polypeptide; a nucleotide encoding an alpha-helical connecting the nucleotide encoding the Cas polypeptide to a nuclear localization signal (NLS); and a second promoter operably connected to a guide RNA (gRNA).