Truncated CRISPR-Cas9 Proteins for AAV Packaging
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Solution Overview
Problem
The large size of Cas proteins, particularly SpCas9, poses a challenge for delivery in genome engineering applications, especially in vivo, as it exceeds the packaging limit of adeno-associated virus (AAV) vectors, limiting customization and addition of genetic elements.
Innovation Solution
Development of polypeptides with specific deletions and missense mutations in CRISPR-Cas proteins, such as Streptococcus pyogenes Cas9, that maintain DNA-binding activity but are optimized for reduced size, enabling efficient packaging and customization within AAV vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the full-length SpCas9 protein is used for genome engineering, then DNA-binding activity and cleavage function are maintained, but the protein size (around 4.2 kb) exceeds the packaging limit of AAV vectors (around 4.5 kb), limiting in vivo delivery and customization
Solution Approach 1:
The patent extracts and removes the HNH nuclease domain (amino acids 775-909) from the full-length SpCas9 protein to create a truncated version (trSpCas9). This extraction eliminates the DNA-cleavage function while preserving the DNA-binding capability, reducing the protein size to fit within AAV packaging limits for in vivo delivery
Solution Approach 2:
The patent segments the SpCas9 protein into functional domains, separating the DNA-binding function (REC domains and PAM interaction region) from the DNA-cleavage function (HNH and RuvC domains). By retaining only the binding-related segments and removing the nuclease domains, the protein is optimized for targeted binding without excessive size
2Adaptability or versatility
If catalytically dead Cas9 (dCas9) is used for DNA targeting without cleavage, then in vivo delivery becomes feasible with AAV vectors, but the packaging capacity is severely constrained with little room for customization and addition of effector domains
Solution Approach 1:
The patent removes the bulky HNH domain and part of the RuvC domain from dCas9 to create a more compact truncated version. This extraction reduces the protein size further, creating sufficient packaging capacity within AAV vectors to accommodate customized effector domains fused to the truncated Cas9, thereby enhancing adaptability and versatility
Solution Approach 2:
The patent changes the structural parameters of the dCas9 protein by deleting specific amino acid sequences (HNH domain and RuvCIII-b). This parameter change reduces the overall protein size while maintaining the essential DNA-binding function, enabling flexibility for future customization with effector domains
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
These optimized polypeptides facilitate site-specific DNA engineering by allowing for the recruitment of effector domains, enhancing the versatility and delivery of CRISPR-Cas systems for biomedical and biotechnological applications.
Implementation Method 1
the element that targets the Cas endonuclease to the correct location in the genome is simply a short piece of guide RNA
Implementation Method 2
Cas proteins from different bacteria will recognize different PAMs in general. Various protein domains are involved in interacting with the DNA
Implementation Method 3
the Cas enzyme can cleave both strands of the DNA upon binding, thereby generating a double-stranded break. The Cas protein contains two nuclease domains, the HNH and RuvC domains, each of which cut opposite strands of the DNA
Data Source
AI summary
The present invention relates to a polypeptide comprising at least one at least one deletion selected from the group consisting of ΔHNH (Δ775-909), ARuvCIII-b (Δ1002-1074), AREC1-a (Δ510-655), AREC1-b (Δ525-587), AREC1-c (Δ662-710), AREC2 (Δ180-308), AREC2-a (Δ212-244), AREC2-b (Δ244-276), AREC2-c (Δ276-308), AREC2-d (Δ199-283), AREC2-e (Δ198-257), AREC2-f (Δ235-286), AREC2-g (Δ217-266), AREC3 (Δ498-712) and combinations thereof, wherein the position numbering is in accordance with SEQ ID NO: 1 encoding for S. pyogenes Cas9, and wherein the polypeptide has CRISPR-Cas DNA-binding activity. The polypeptide may further comprises a missense mutations selected from G12R, T13K, T13R, N14K, N497K, T657K, T657R, N767K, T770K, T770R, Q920K, Q920R, S1109R, D1135K, D1135R, S1338R and combinations thereof. Also claimed are nucleic acid molecules encoding for said polypeptides, compositions and method of site-directed engineering of a target DNA thereof.